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THE AMERICAN JOURNAL

OF

ANATOMY

MANAGING EDITOR

CHARLES R. STOCKARD
Cornell University Medical School

ASSOCIATE EDITORS

CLARENCE M. JAcKSON Haroup D. SENIOR
University of Minnesota New York University

Henry Mck. Knowrr GEoRGE L. STREETER
University of Cincinnati Carnegie Institution

JANUARY, 1922—JULY, 1922

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA, PA.

CONTENTS

No. 1. JANUARY

Rospert H. Bowen. On certain features of spermatogenesis in amphibia and insects.

IAN WiO) TOVEHIGSH GIO NEN CIS bea 1kea DI) eae GMP Dina eeu Game no acleocu sete dormers ce cilioerc oer 1
E. V. Cowpry. The reticular material as an indicator of physiologic reversal in secretory
polarity in the thyroid cells of the guinea-pig. Two plates (fourteen figures).... 25

WarRREN H. Lewis. Endothelium in tissue cultures. Five plates (twenty-four figures). 39

Otto F. KaMpMEIER. The development of the anterior lymphatics and lymph hearts in
anuran embryos. Thirty-five figures including eight colored plates................ 61

Hautsty J. Baga. Disturbances in mammalian development produced by radium
emanation. Ten text figures and three plates (figures eleven to fifteen)............ 133

No. 2. MARCH

Epwarp A. Boypen. The development of the cloaca in birds, with special reference
to the origin of the bursa of Fabricius, the formation of a urodaeal sinus, and the
regular occurrence of a cloacal fenestra. Forty-one figures.....................-..- 163
Ivan E. Watirn. On the nature of mitochondria. I. Observations on mitochondria
staining methods applied to bacteria. II. Reactions of bacteria to chemical treat-

Pole Ta he CMEC PUBIC (RUIN G) AEUIT CS). cs 51%. sido ovesc 5. 5,30 51.0e A ae OPS Pao roe oes crete a arabes 203
BEATRICE WHITESIDE. The development of the saccus pedal mphaticus i in Rana tem-
Pekar a oiiies Nineteen) HP WEES .f.x.5\iuto.s <x» alaie's ate eaaees oP wh trerasn ht eso wes 231
Ne-'3.. - NDA.
Hersert G. Witutson. The terminals of the human bronchiole. Nine figures.......... 267
Epagar ALLEN. The oestrous cycle in the mouse. Twenty-four figures................ 297
BraDLey M. Patten. The formation of the cardiac loopin the chick. Two text figures
PRR RER CELL CEI oh enee asf erry oil aya ca: a.al's-<) «01 ara SNe epee eta e «= oben evniae RMR eyes 373
No; 4.. JULY
R. S. CunnineHam. The reaction of the cells lining the peritoneal cavity, including the
germinal epithelium of the ovary, to vital dyes. One plate (eight figures).......... 399
Raymonp M. Setue. Changes in the vaginal epithelium of the guinea-pig during the
Genmous cycle. Dhree: plates (eleven figures)... 6.6. 008s oes ce eee sk weew ac cpin soe 429
ui

lv CONTENTS

Ivan E. Wauuin. On the nature of mitochondria. III. The demonstration of mito-
chondria by bacteriological methods. IV. A comparative study of the morphogenesis
of root-nodule bacteria and chloroplasts. Two plates (nine figures)............... 451
A. W. Bretuamy. Differential susceptibility as a basis for modification and control of
development in the frog. II. Types of modification seen in later developmental
stager. ‘sS¢eventy steures Kat eae as eis he ae a eaiGr.\feialtns.' Jo eae 473
A. L. Sauazar. Sur une forme particuliére d’atrésie des follicules de deGraaf (lapine),
révélée par la méthode tannoferrique. Cinq figures................ ee eee e ee eee eens 503

Resumen por el autor, Robert H. Bowen

Sobre ciertos rasgos de la espermatogénesis de los anfibios e
insectos

Un estudio comparativo de las espermatidas de Plethodon,
Lixus, Rhomaleum y Ceutophilus ha confirmado la opinién de
que el acrosoma del espermatozoide se origina en relacién con el
aparato de Golgi. En Plethodon se ha demostrado que el apa-
rato citado y el idiozoma, después de producir el acrosoma, son
expulsados y pasan a la base de la cabeza del espermatozoide
donde puede observarse durante algin tiempo. Nunca pre-
senta una connexién orgdnica con los centriolos situados en su
vecindad, ni tampoco juega ningtin papel en la formacién del
segmento intermedio del espermatozoide, conforme se ha su-
puesto. En Lixus y Ceutophilus el acrosoma se origina de un
modo muy semejante al de los hemfpteros. En el saltamontes
Rhomaleum, por otra parte, los corptisculos de Golgi de la es-
permitida nunca se fusionan para formar un solo acroblasto
como sucede en las formas precedentes, sino que cada elemento
de Golgi parece contribuir una porcién individual y separada,
formandose el acrosoma en virtud de la fusién de estas numero-
sas partes.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 27

ON CERTAIN FEATURES OF SPERMATOGENESIS IN
AMPHIBIA AND INSECTS

ROBERT H. BOWEN
Department of Zoology, Columbia University
TWO PLATES (FORTY-SIX FIGURES)

In a recent paper (Bowen, ’20) on insect spermatogenesis I was
able to demonstrate a relationship between the acrosome and the
Golgi apparatus, and from this and facts previously published by
other workers, I suggested that ‘‘The acrosome of the animal
sperm is probably universally formed in connection with the
Golgi apparatus, and has nothing to do with spindle fibers or
other cell parts.’? At that time the evidence from other groups
was very scanty indeed, the work of Sjoevall, Schitz, Gatenby,
and Duesberg on various molluscs and mammals furnishing
almost the only indications of the correctness of this suggestion.
On the other hand, there were several well-known cases in which
the current descriptions seemed to present serious difficulties, if
not a direct contradiction. | decided, therefore, to examine
some of these cases more carefully, together with material from
other groups in which the Golgi apparatus had not yet been
studied. In working up the material, a number of unexpected
features were brought to light and it seemed of value to look
more carefully into some of the cytoplasmic constituents which
had not been accurately followed heretofore. This, however,
called for the collection of much more material and further experi-
menting with fixation and staining—tasks which could not be
immediately undertaken. I have accordingly decided to pub-
lish the major facts relating to acrosome formation, together with
certain other features of interest in the spermatogenesis of several
Amphibia and Insecta, leaving the details for a later study. A
review of technical methods and acknowledgment of much assist-
ance in the collection and identification of material will be for

the present postponed.
1

2 ROBERT H. BOWEN

AMPHIBIA

Accounts of the formation of the acrosome and the middle-
piece in the urodele sperm have long presented a most puzzling
contradiction. According to Meves (’97), in Salamandra, the
acrosome (‘Sphaerenblaeschen’) arises by the fusion of many
small, clear vesicles which appear in the idiosome (‘Sphaeren-
substanz’) of the spermatid. The vesicular acrosome thus
formed becomes the apical body of the sperm, while the remnant
(‘der kleine Rest’) of the sphere substance disappears completely.
The ‘middle-piece,’ on the other hand, is formed by the proximal
centriole alone, which penetrates the nuclear membrane and thus
comes to lie inside the nucleus. But McGregor (’99), who has
made a similar study of Amphiuma, gives a quite different inter-
pretation of these details. According to McGregor, the acro-
some arises from the sphere (idiosome) in a manner essentially
similar to that described by Meves, except that the remnant of
the sphere does not disintegrate. Instead it migrates with the
centrioles to the opposite pole of the nucleus, where it forms the
major part of the middle-piece, in which the small, spherical
proximal centriole becomes embedded. McGregor also describes
the penetration of the middle-piece into the nucleus, both authors
thus agreeing that the middle-piece is intranuclear. With re-
spect to the fate of the sphere remnant (Golgi remnant of my
nomenclature) and the origin of the middle-piece, there was thus
a discrepancy which has never been cleared up. It was with the
idea of ascertaining the true relations of the Golgi apparatus
(plus idiosome), acrosome, and centrioles that this work was
undertaken. The material for this study has been drawn from
a single species, Plethodon cinereus Green, though I hope eventu-
ally to examine other urodeles in a comparative way.

The cytoplasmic structures so far made out in the primary
spermatocytes are of at least three categories, the Golgi appa-
ratus-idiosome complex, the mitochondria, and a number of
darkly stained granules of doubtful nature, but possibly to be
considered as chromatoid bodies. The mitochondria, present
in rather limited amount, occur as more or less scattered rodlets
of very small diameter, similar to those figured in Geotriton by

SPERMATOGENESIS IN AMPHIBIA AND INSECTS 3

Terni (14). The idiosome and Golgi apparatus have been often
described by earlier workers. Together they form much the
most conspicuous object in the spermatocytes. The idiosome
appears as a more or less irregularly spherical mass, staining
rather more darkly than the general cytoplasm. ‘To its surface
are applied a large number of small, separate Golgi rodlets, each
shaped somewhat like a banana (fig. 1), and seemingly never
interconnected to form a ‘network.’ These rodlets, now defi-
nitely shown to be Golgi elements, have of course been long known
under a variety of names, e.g., the ‘Archoplasmaschleifen’ of
Hermann, the ‘Centralkapsel’ and ‘Pseudochromosomen’ of
Heidenhain, and the ‘formazioni periidiozomiche’ of Terni.

During the maturation divisions the idiosome goes to pieces
and its exact behavior during the division period is unknown.
The Golgi rodlets undergo a great loss in staining capacity, and
appear to be simultaneously fragmented, so that with the usual
methods they cannot be satisfactorily followed in the division
stages. It is certain, however, that the Golgi pieces (dictyo-
somes) are collected about the poles of the spindle (fig. 2) as
described in invertebrates by several workers, although the exact
method by which this distribution is achieved has not yet been
clearly made out. At the close of the first maturation division
the idiosome is reconstituted with the Golgi rodlets applied to its
surface as before (fig. 3). Similarly, in the spermatids the same
structure is built up soon after the second maturation division
is completed. In my preparations, however, the Golgi rodlets,
as distinct elements, are soon lost, and the outer part of the
acroblast (as the Golgi apparatus plus idiosome may now be
called) tends to impregnate rather uniformly with the various
Golgi methods (figs. 5 and 6).

The acroblast lies at first some little distance from the nucleus;
but presently it moves over into contact with the nuclear mem-
brane and a small clear vesicle makes its appearance on one side
of the acroblast (fig. 5). This is the “Sphaerenblaeschen’ of Meves
and the acrosome of McGregor. In general appearance it closely
resembles the homologous structure which I have described in
the hemipteran spermatid (Bowen, ’20), and for which I have

r

4 ROBERT H. BOWEN

adopted the term acrosome. ‘The acrosome increases in size,
becoming a large, clear vesicle, to the surface of which the remains
of the acroblast are attached (figs. 6 and 7). The centrioles pres-
ently become conspicuous, being sharply stained with Fe-hema-
toxylin. They are always located in the neighborhood of the
acroblast (figs. 6, 7, and 8); indeed, they seem often to be resting
on its surface. The relation seems, however, to be purely a
topographical one.

When the acrosome has reached a certain stage, it becomes
applied closely to the nuclear membrane, and gradually the cell
wall is drawn down over it, creating the impression that the
acrosome has actually protruded through the cell wall (fig. 8).
Throughout this period of fixation, the acroblast remains close
to the acrosome (figs. 8 and 9), but the connection between the
two has obviously become very loose. In fact, the acroblast
or, as it may now be called, the Golgi remnant, becomes more or
less spread out or broken up into separate masses (fig. 9),
and in Fe-hematoxylin preparations is usually not to be made
out at all. This loss in staining capacity is obviously the
source of Meves’ erroneous description of the disappearance of
the ‘Sphaerensubstanz.’

The Golgi remnant, together with the centrioles, lingers for a
time in the immediate vicinity of the acrosome, and then both
are shifted to the opposite pole of the nucleus establishing the
future long axis of the sperm. Everything indicates that
McGregor was correct in holding that the separation of acrosome
and centrioles is due to the active migration of the centrioles,
rather than of the acrosome, as Meves contended.

The spermatid nucleus meanwhile becomes very irregular in
shape and then begins to draw out to form the head of the sperm
with the acrosome at one end and the centrioles at the other
(fig. 11). The Golgi remnant lies in the cytoplasm at the base
of the head close to the centrioles (figs. 11 and 12), becoming
oftentimes more or less broken up and diffuse. It is easily
demonstrated by the special methods for the Golgi apparatus,
but not by the usual Fe-hematoxylin techniques. The Golgi
remnant remains in this same position for a long time; indeed,

SPERMATOGENESIS IN AMPHIBIA AND INSECTS 5

long after the stage shown by McGregor in his figure 32, in which
it has supposedly been incorporated with the proximal centriole
to form the middle-piece of the sperm. Its ultimate fate is
unknown, preparations of late stages in sperm formation not yet
having been successfully impregnated. Meanwhile the proximal
centriole (in particular) increases progressively in size and stain-
ing capacity (compare figs. 11, 12, and 18) and becomes applied
very closely to the nuclear membrane. At first it is spherical,
but as the head elongates and becomes narrower, the sphere
begins to draw out (fig. 14) and it becomes eventually a long rod
(fig. 19)—the (centrosomal) middle-piece of the sperm. In this
process I have been quite unable to find any evidence of an
actual penetration of the nuclear wall by the ‘middle-piece,’ as
described by both Meves and McGregor. The centriole seems
rather to be merely in close contact with the nucleus, as is the
case in many other animal sperms.

With respect to the origin of the middle-piece, I am, therefore,
in general agreement with Meves. The unusual topographical
relation existing between the Golgi remnant and the centrioles
was doubtless the cause of McGregor’s error in the derivation of
the middle-piece, coupled also with the poor staining conditions
at this period. There is also a possibility that some large, darkly
staining granules of doubtful relations, which occur in the sper-
matids, may have been confused by him as derivatives of the
cast-off ‘sphere.’

Finally, the conditions in the completed middle-piece described
by McGregor remain to be explained. Since, according to this
author, in the formation of the middle-piece the proximal cen-
triole merely becomes embedded in a mass of sphere material,
it should be possible to separate these two components by dif-
ferential staining. This was in fact apparently accomplished
(McGregor’s fig. 32), the sphere material staining less heavily
than the centriole. My own observations of the proximal cen-
triole (so-called) suggest, however, a quite different interpre-
tation of this appearance. As was noted by McGregor, the
proximal centriole in the earlier spermatid stages often appears

6 ROBERT H. BOWEN

bipartite or dumb-bell shaped,! the two parts being typically
equal in size. Meves figures much the same thing, though he
described the formation as a curved rod. This same division of
the proximal centriole occurs in Plethodon (figs. 7 and 10), the sep-
aration of the two parts being sometimes very marked. ‘To one of
the halves the axial filament is attached. When the nucleus begins
to draw out, this division of the proximal centriole is apparently
lost in many cases—sometimes, no doubt, by the recombination
or fusion of the two parts and at other times by the orientation
of the centrioles. At the same time, the part to which the axial
filament is attached increases very rapidly in size, while the other
part remains relatively small and inconspicuous. However,
with a little searching, examples can be found at any of the
intermediate stages in which the two parts of the proximal cen-
triole can be clearly separated (figs. 18, 14, and 15). That the
two bodies in these cases are actually the equivalent of the appar-
ently single proximal centrioles of other cases can be demon-
strated by comparison of adjacent spermatids in which the parts
can or cannot be separated (figs. 138 and 14). It seems probable,
as Professor Wilson has suggested to me, that in McGregor’s
preparations the large centriole was extracted more than the
smaller one, giving rise to the interpretation already noted. The

1 It should be pointed out that the origin of this bipartite condition has not
yet been adequately worked out. I have followed the usual custom in considering
the two parts as formed by the division of the proximal centriole, my own prepa-
rations not permitting a detailed study of the very early spermatid centrioles.
Doctor Wilson has, however, called my attention to the fact that generally in
the differentiation of the sperm a ring arises from only a portion of the distal
centriole, while the remainder later comes into close relation with the proximal
centriole. It is possible that something of the same kind obtains in the case of
the urodele. Thus we might consider the portion of the so-called ‘proximal’
centriole to which the axial filament is directly connected as the central part of
the ring centriole, while the other portion would actually be the proximal centriole
itself. This would explain the apparent transfer of the insertion of the axial
filament from the distal to the proximal centriole as required by McGregor’s
description, and would fall in line with the usual accounts of sperm formation
which derive the axial filament from the distal centriole. Further study of this
possibility is contemplated. However, in view of the uncertainty attaching to
these speculations, I have thought it best to follow the traditional accounts for
the present purely as a matter of convenience in description.

SPERMATOGENESIS IN AMPHIBIA AND INSECTS r

variability in staining behavior of the centrioles in Plethodon
leads me to look upon this as a very plausible explanation.

In the late stages this small centriole seems to fuse with the
free end of the much elongated middle-piece proper, from one
end of which the axial filament arises (compare fig. 15). The
function of this small centriole is not yet understood, but a pos-
sible explanation has suggested itself, which I hope eventually
to prove or disprove definitely. It will be recalled that the tail
of the urodele sperm bears a long, undulating membrane, the
base of which is inserted along the axial filament of the tail,
while the free edge is marked by a definite filament somewhat
smaller in size than the main tail filament. The axial filament
proper undoubtedly arises from the large centrosomal middle-
piece. The exact origin of the membrane filament has not,
however, been satisfactorily explained. I would like to suggest,
on the basis of observations already made, the possibility of the
origin of this filament from the second, eventually much the
smaller, of the original halves of the proximal (?) centriole. Un-
fortunately, this filament is so delicate that its free portion can
be made out in its earlier stages only with some difficulty, while
the insertion can never be satisfactorily followed. It frequently
happens, however, that the developing sperms are cut obliquely
through the base of the head, and in such sections one can some-
times see what appears to be a fine filament arising from the
small centriole and passing backward through the ring centriole
(figs. 16 and 17). (See also fig. 18, in which the ring was not
included in the section.) Should further study bear out such an
interpretation, the origin of the membrane filament would fall
in line with the facts already known as to the origin in general
of vibratile filaments. It is possible, nevertheless, that the
small centriole plays a réle in the late stages during the drawing
out of the ring centriole; for I have constantly observed a small
darkly staining mass at the base of the tail filament which seems
to originate from the centrosomal middle-piece (fig. 19). This
mass subsequently moves out along the filament toward the free
end of the elongating ring centriole, but its fate and function are
alike unknown.

8 ROBERT H. BOWEN

I have also found in the spermatids a collection of granules
which seem to be preserved best in the absence of acetic acid.
During the formation of the acrosome these granules are arranged
in a circle around the acroblast and lie close to the nuclear
membrane. This arrangement is a constant and striking one
(fig. 4). The nature and fate of these granules are not known.
Possibly they are related to the chromatoid bodies of other
animals. They may be related to the granules previously men-
tioned as occurring in the spermatocytes. A more critical study
of these granules will be undertaken on new material.

Summary

1. The acrosome of the urodele sperm arises as in many other
animals from the Golgi apparatus plus idiosome (acroblast),
which is later cast off and remains for some time in the cytoplasm
near the base of the sperm nucleus.

2. The middle-piece of the sperm is derived from the so-called
proximal centriole, and is not, as McGregor claimed, “chiefly
derived from the remnant of the sphere.”’

3. The origin of the filament on the free edge of the undulating
membrane of the sperm tail from one part of the originally bipar-
tite proximal (?) centriole is suggested as a possibility.

COLEOPTERA

My observations on the Coleoptera are confined to a single
species, Lixus concavus Lec., which was examined merely to
check up the origin of the acrosome in the light of my previous
work on Hemiptera. In this beetle, the Golgi apparatus is pres-
ent in the spermatocytes in the form of numerous scattered
Golgi bodies (fig. 27) similar in a general way to those which I
have described in the pentatomids. I have not followed out the
preliminary history of these Golgi bodies or their distribution in
the maturation divisions. In the early spermatids, however,
before the mitochondria have condensed to form the nebenkern,
the Golgi bodies are easily demonstrated, scattered irregularly
in the cytoplasm (fig. 28). They gradually draw together (fig.
29), and eventually fuse to form a single mass (fig. 30), the acro-

SPERMATOGENESIS IN AMPHIBIA AND INSECTS 9

blast, exactly as in Hemiptera. ‘This lies at first in the angle
between nucleus and nebenkern, whence it moves to an anterior
position (fig. 30). Later it appears again at the base of the
head (figs. 31 and 34), so that it probably undergoes a migration
similar to that which occurs in Hemiptera. The acroblast in
this beetle impregnates in a very peculiar manner. Instead of
forming a thimble-shaped mass with a heavily impregnated
periphery as in the pentatomids (Bowen, ’20), it is apparently
shaped like a disc, only the periphery of which is blackened by
osmic acid. That this is a ring, not an optical section of some
solid surface, is easily demonstrated by viewing the acroblast
from different angles (compare figs. 30 and 31).

From the acroblast the acrosome arises by a process of dif-
ferentiation similar in all probability to that in the Hemiptera
and Amphibia. In this case, however, the acrosome is so very
small that it cannot be satisfactorily demonstrated until the time
approaches for the casting off of the acroblast, when it can be
made out as a small vesicle applied closely to the nuclear mem-
brane (fig. 36). Then the acroblast separates from the acro-
some proper (figs. 37 and 38), and passing rapidly back along
the tail (fig. 39), remains visible as a conspicuous blackened ring
for a considerable period. Its fate is doubtless similar to that
in the Hemiptera. At the time the acroblast is cast off, the head
of the sperm is usually bent rather sharply over, so that its major
axis is nearly at right angles to that of the tail filament. It is
accordingly difficult to say whether the acrosome is deposited
directly in place and becomes subsequently anterior by the
straightening out of the head; or whether there is an active migra-
tion of the acrosome to its definitive position, as in the penta-
tomids. Probably both factors play a part. At all events, the
head eventually straightens out, and the acrosome can be made
out as a minute body applied to its tip (fig. 40). Subsequently
the head becomes much drawn out, as in other insect sperms.

I have also been able to make some observations on the neben-
kern which form an interesting supplement to my recent dis-
cussion of this subject (Bowen, ’22b). In Lixus the nebenkern
goes through a process of condensation quite similar to that

10 ROBERT H. BOWEN

which I have described in Brochymena, and already outlined in
part by Shaffer (17) in Passalus, and Holmgren (’02) in Silpha.
The chromophilic substance (Bowen, ’22b) seems to form a
plate-work very like that in the Hemiptera, and I can find no
trace whatever of a ‘spireme’ such as Gatenby (718) claims to
have demonstrated in Tenebrio. At all events, in the later
stages of condensation, the chromophilic substance is arranged
exactly as in the pentatomids, and cross-sections of the neben-
kern (figs. 32 and 33A and B) could hardly be distinguished
from similar sections of Brochymena. (Compare Bowen, ’22 b,
figs. 21A and B, also fig. 13.) In one respect, however, there is
a fundamental difference in the behavior of the nebenkern. In
the Hemiptera the chromophilic substance disappears completely
and the nebenkern is-divided into two parts during the initial
stage in its drawing out; while in the Coleoptera the drawing out
of the nebenkern has progressed much farther before these same
results are attained. Thus, in figure 34, a stage in the final con-
densation of the chromophilic substance is shown, which is quite
comparable to my figure 16 (Bowen, ’22b) of Brochymena;
while in an adjacent spermatid (fig. 35) the chromophilic sub-
stance has completely (?) disappeared.

In the paper already referred to, I attempted to show that the
division of the nebenkern always waits on the final disappearance
of the chromophilic substance. The observations of Holmgren
on Silpha indicated that this was the case in the beetles, but
Shaffer’s account of Passalus left the point somewhat in doubt.
I have, therefore, reexamined this point more carefully in Lixus,
and I find the facts exactly as described by me in the Hemiptera.
Cross-sections of the nebenkern through the chromophilic sub-
stance and also above and below it demonstrate conclusively
that the nebenkern is divided only in the region from which the
chromophilic substance has been withdrawn (figs. 833A and
B, cross-sections of the nebenkern at a stage approximately like
fig. 34. Compare these cross-sections with figs. 214 and B from
Brochymena, Bowen, ’22b).

The halves of the nebenkern ultimately spin out to form the
tail sheaths so characteristic of insect sperms, and I can find no

SPERMATOGENESIS IN AMPHIBIA AND INSECTS 1a

evidence of a degeneration of the mitochondrial elements such
as Holmgren described in Silpha. The central substance (Bowen,
22 b) was not demonstrable in my preparations, so that I have
been unable to check Holmgren’s account of this feature.

Summary

1. The acrosome arises in connection with the Golgi apparatus
plus idiosome, in a manner similar to that described in several
other animals.

2. The nebenkern passes through a series of condensation
phenomena similar to those of the Hemiptera, and is completely
divided only with the disappearance of the chromophilic sub-
stance.

ORTHOPTERA

The development of the acrosome in various groups of Orthop-
tera has been studied by many workers with very discordant
results, a short résumé of which I have given in another place
(Bowen, ’22a). Most of these cases, however, have been easily
interpreted on the basis of conditions found in the Hemiptera,
but in the case of the grasshopper the facts are as yet by no
means clear. It was with the hope of clearing up the whole
problem of the acrosome in Orthoptera that this study was under-
taken. Thus far I have had opportunity to examine only a few
species from the families Acrididae and Tettigoniidae.

Family Acrididae

Although the grasshoppers have been studied by many workers
there has been a uniform failure to make out any details what-
ever concerning the acrosome. Indeed, Buchner seems to have
been the only one who even noted its occurrence. According to
his latest account (Buchner, ’15), the acrosome is derived from
the ‘proximal’ centriole by a process of division—an origin which
is obviously quite different from that to be expected on the basis
of my hypothesis. As a matter of fact, Buchner failed to make
out most of the essential stages, due to the fact that the cyto-

12 ROBERT H. BOWEN

plasmic elements in the grasshopper seem to be most difficult to
preserve and stain satisfactorily. My observations thus far are
based on two species, Rhomaleum micropterum Beauy., and
Dissosteira carolina Linn.

The Golgi apparatus in the grasshoppers is present in the
primary spermatocytes in the form of many scattered Golgi
bodies not essentially unlike those which I have described in the
Hemiptera. The division stages have not yet been worked out,
but at the close of the second maturation division the Golgi
elements can be demonstrated clearly, being scattered about in
the cytoplasm around the nucleus and mitochondria (fig. 20).
The latter consist of a group of rather imperfect threads which
have been derived from the second maturation division in a man-
ner somewhat similar to that noted in Hemiptera. These threads
soon condense to form the typical spherical nebenkern of insects.

The separate Golgi bodies are not easily demonstrated with
clearness, but it is certain, especially from later stages, that each
one is made up of two substances, one of which is more readily
stained than the other which it partially encloses. (Compare
with the Hemiptera which I have described fully (Bowen, ’22 a).)
In one respect, however, the Golgi bodies differ in behavior very
decidedly from other cases which have been described. ‘They
remain separate for the most part as distinct bodies (figs. 23 and
24), and never fuse to form the massive acroblast so character-
istic of the animal spermatid. Occasionally, it would appear,
two or three fuse together to form a larger aggregate, but in
general this does not occur. These Golgi bodies tend now to
collect near the nuclear membrane, particularly on one side of
the nebenkern (figs. 21 and 23), and they remain, generally
speaking, in this vicinity until the nebenkern begins to elongate
(fig. 24). Then they begin to migrate back along the tail (fig.
25), and are probably cast out in the protoplasmic mass sloughed
off the tail at the close of sperm formation.

In the later spermatid stages and especially just prior to the
backward migration of the Golgi bodies, an intensely staining glob-
ule can be made out in contact with the nuclear membrane not
far from the centriole (figs. 23 and 24). This is at first difficult to

SPERMATOGENESIS IN AMPHIBIA AND INSECTS 13

distinguish with certainty, but it seems to increase in size, and
when the Golgi bodies clear away from the nucleus it is a very
conspicuous object. This globule is the acrosome. It now
migrates around to the opposite side of the head (nucleus) (fig.
25), always retaining its close contact with the nuclear wall. As
it moves it becomes differentiated into a basal, plate-like portion
which rests on the nucleus, and a small knob directed toward
the cell wall (fig. 25), the whole reminding one of a collar-button.
As the head begins to elongate in the manner characteristic of
the insect sperm, the basal portion of the acrosome becomes
drawn out into two rod-like lugs which extend back over the
surface of the nucleus for a short distance and apparently serve as
a means of anchorage for the acrosome (fig. 26). The distal
portion becomes drawn out as a little ball on the end of a short
rodlet. Very frequently this can be divided into two—a duplex
condition which is possibly the characteristic one, though visible
only when the sperm head is favorably turned. As the head
draws out, the acrosome retains, for a time, this rod-and-ball
structure; its ultimate history has not been studied.

Unfortunately, the exact method by which the acrosome is
produced has not been made out. The separate Golgi bodies
are themselves so small that in my preparations I was not able
to follow the history of each individual. It seems probable, how-
ever, that from each one is differentiated its small proportionate
share, and by the deposition of many such parts the acrosome is
gradually built up. Instead of the Golgi bodies’ fusing to form
a single acroblast from which the acrosome is differentiated in
toto as in most animals, each Golgi body is an acroblast in itself
and the acrosome arises as a fusion product of the portions con-
tributed ‘by each such ‘acroblast.’ This all fits in with the facts
which I have made out in the Hemiptera. In the pentatomids
I was able to show that occasionally the Golgi elements fail to
fuse on time, and in such cases each partial acroblast produces
its own acrosome of a size proportionate to the available material
(Bowen, ’22a, fig. 54). Thus, what may be an occasional
accident in the bug becomes in the grasshopper the customary
procedure.

14 ROBERT H. BOWEN

In addition to these observations on the acrosome, I have been
able also to add some facts corroborative of the nebenkern history
which I have described in the Hemiptera (Bowen, ’22 b). Thus
I have found that the nebenkern passes through a process of
differentiation, which, in the beginning at least, is strikingly like
that in Brochymena, and I have been unable to make out the
thread-like formation figured by Giglio-Tos and Granata (’08).
As in the Hemiptera, the chromophilic substance gradually con-
denses and eventually disappears entirely, figure 21 being a
cross-section of the nebenkern just before its final disappearance.
Following this, the nebenkern divides. The condensation of
the chromophilic substance in the grasshopper nebenkern takes
place so rapidly that the nebenkern divides some time before it
begins to elongate, the two halves rounding up in a very charac-
teristic manner (fig. 23). Another case is thus added to those
already described, which show that the complete division of the
nebenkern follows immediately upon the disappearance of the
chromophilic substance. With respect to the condition of the
chromophilic substance at the time the nebenkern begins to
elongate, an interesting series is thus formed by the grasshopper,
the bug, and the beetle (and possibly the butterfly).

Finally, in Cajal preparations, I was able to demonstrate in
the nebenkern (as in the Hemiptera) the occurrence of a material
which I have called the ‘central substance.’ This stuff in the
grasshopper nebenkern is by no means as regular in its arrange-
ment as in Kuschistus, for example. Instead, it forms a very
indefinite mixture of granular (and thread-like?) elements which
combine to make an exceedingly complicated picture (fig. 22).
When the nebenkern halves elongate this central substance is
drawn out also, and seems eventually to form a delicate thread-
like core for the mitochrondrial tail sheaths. The vesicles which
I have described on the tail sheaths in Hemiptera appear like-
wise in the grasshoppers, but are by no means so conspicuous.
Their general history seems not unlike that in the Hemiptera.

SPERMATOGENESIS IN AMPHIBIA AND INSECTS 15

FAMILY TETTIGONIIDAE

In contrast to the unusual method of acrosome formation
found in the grasshoppers, among many of their relatives the
process would appear to be a very simple one, quite like that in
the Hemiptera. Thus far I have examined carefully only Ceu-
thophilus maculatus Harris, but the condition in this form is
probably similar to that in other crickets and the locustids,
where the published accounts are very unsatisfactory indeed
(see Bowen, ’22 a, for a short résumé). In Ceuthophilus I have
not yet obtained satisfactory Golgi preparations of the sperma-
tocytes and earliest spermatid stages. However, during the
early steps in the condensation of the chromophilic substance,
the acroblast, in the form of a single compact sphere, appears in
the cytoplasm in its accustomed location in the angle between
nucleus and nebenkern (fig. 41), indicating an origin from the
fusion of scattered Golgi elements as in the beetle and the bug.

The differentiation of the acrosome begins immediately, and
a small vesicle soon appears in connection with the acroblast
quite as in the Hemiptera (fig. 42). The vesicle is sometimes
clear and transparent (fig. 48), but very frequently it takes the
stain intensely (fig. 42), by which it is rendered more conspicuous.
I have noticed a similar tendency in Murgantia after certain
staining technique. The position of the acroblast-acrosome com-
plex with reference to the nucleus is not constant as in the Hemip-
tera, and the acrosome often does not touch the nuclear wall
at all.

Shortly before the nucleus begins to elongate to form the sperm
head, the acrosomal vesicle becomes applied to the anterior
surface of the nucleus (fig. 43), the acroblast still maintaining
its original attachment to the acrosome. Presently the acro-
some flattens out on the surface of the nuclear membrane (fig.
44), and its connection with the acroblast is severed (fig. 45).
The latter quickly migrates to the base of the head (fig. 46) and
thence moves backward along the tail exactly as in other insects,
in all of which its ultimate fate is probably the same. The acro-
some, already in its definitive position, rounds out to form a

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 1

16 ROBERT H. BOWEN

knob-like apical piece (figs. 45 and 46), the further history of
which has not been studied.

Aside from the mitochondria, the spermatids of Ceuthophilus
contain two cytoplasmic components of interest. The first of
these appears in the early spermatids as a small flocculent mass,
more or less granular in texture and blackening with osmie acid
(modified Kopsch method) (fig. 41). Later this mass seems to
move backward along the tail (fig. 42), but it no longer impreg-
nates clearly and cannot be followed satisfactorily. It seems to
correspond to the granular mass of similar behavior which I
have described in the Hemiptera under the name of spermatid
remnant (Bowen, ’22 a). In Ceuthophilus, however, this mate-
rial seems to be traceable at least back to the maturation divi-
sions, and a more complete study of its history and significance
is now being attempted.

The other cytoplasmic component referred to above is a body
apparently homologous with the ‘formation juxta-nucléaire’
described in Gryllotalpa by Voinov (16). According to this
author, the late primary spermatocytes contain four of these bodies
which are distributed to the spermatids by a regular division
process, each spermatid always receiving one juxtanuclear body.
The last contention seems to hold true in Ceuthophilus, but I
have been unable thus far to substantiate the earlier history as
outlined by Voinov, and in my preparations this body first
becomes clearly demonstrable in the differentiating spermatids.
In shape, Voinov describes it as a flattened, oval body the peri-
phery of which stains very darkly. In Ceuthophilus also it has
the same disc-like shape, but whether the whole periphery stains
heavily (after Benda and various Fe-hematoxylin methods) or
only a portion of it, giving rise to a crescentic appearance, is not
certain (figs. 48 and 45). It is clear from my figures, and in
agreement with Voinoy, that the juxtanuclear body has nothing
to do with the formation of the acrosome. It finally passes out
along the tail together with the Golgi remnant, with which it
probably shares a similar fate. As to the nature of this body,
nothing definite can be stated. Its fate recalls that of the chro-
matoid body, but in other respects there is little ground for com-

SPERMATOGENESIS IN AMPHIBIA AND INSECTS ily

parison. Possibly it is related to the micro-mitosome described
by Gatenby (17) in Lepidoptera. Although we lack any defi-
nite knowledge as to what this juxtanuclear body may be, atten-
tion may nevertheless be called to the fact that it seems to be in
no way related to the Golgi apparatus. The suggestion of
Duesberg (20) (based on Voinov’s account), that these bodies
represent Golgi elements, is, therefore, almost certainly incorrect.
Finally, a word may be added concerning the mitochondria
in the spermatid, especially in view of the wide divergence of
Vejdovsky’s account (in a related genus, Diestrammena) from
more recent work on other insects. In the first place, the
development of the vacuoles on the periphery of the nebenkern
(fig. 41), as described by Vejdovsky, is obviously the first step
in the condensation of the chromophilic substance as already
noted in the Hemiptera, etc. (Bowen, ’22). The chromophilic
substance gradually condenses (fig. 42), though always in a some-
what irregular way, and eventually disappears during the early
stages in the elongation of the nebenkern. The exact nature of
the ‘pattern’ which it forms is not clear in my preparations.
The central substance, correctly described by Vejdovsky in the
intermediate stages (see his figures 182, 183, and 184), seems not
to arise from the chromophilic material as he believed, but to be
differentiated de novo in the unstained area of the nebenkern,
as I have described in the pentatomids and the grasshopper.
The formation of vesicles on the mitochondrial tail sheaths
is very striking in Ceuthophilus, and due to the general failure
of the chromophobic substance to stain, it is not easy to follow
their differentiation. Hence the error of Vejdovsky in describing
them as fatty degeneration products of the central substance, and
I can find no support whatever for his assertion that the mito-
chondrial structures are entirely lost from the sperm. The vesi-
cles in Ceuthophilus are, it is true, very confusing if one has not
become familiar with them in more typical cases, for the inter-
vening portions of the sheaths are often not demonstrated (fig.
43), and one would be readily misled as to the actual meaning of
the vesicles themselves. Somewhat later stages leave little
doubt that the sheaths actually spin out along the tail filament

18 ROBERT H. BOWEN

as in Hemiptera (Bowen, ’22 a, fig. 132). During the early
development of the tail vesicles the central substance can be
demonstrated with Fe-hematoxylin and it can be seen to retain
its threadlike distribution even in the region of the vesicles. It
would accordingly seem that the central substance cannot play
the rdle in the development of the vesicles which the conditions
in Hemiptera led me to suggest as a possibility (Bowen, ’22 b).

LITERATURE CITED

Bowen, R. H. 1920 Studies on insect spermatogenesis. I. The history of the
cytoplasmic components of the sperm in Hemiptera. Biol. Bull.,
vol. 39.
1922 a Studies. II. The components of the spermatid and their
role in the formation of the sperm in Hemiptera. Jour. Morph.
(In press.)
1922 b Studies. III. On the structure of the nebenkern in the
insect spermatid and the origin of nebenkern patterns. Biol. Bull.
(In press.)

Bucuner, P. 1915 Praktikum der Zellenlehre. Berlin.

DurssBerG, J. 1920 Cytoplasmic structures in the seminal epithelium of the
opossum. Carn. Inst. of Washington, Cont. to Embry., no. 28.

GaTreNBY, J. B. 1917 The cytoplasmic inclusions of the germ cells. Part I.
Lepidoptera. Quart. Jour. Mie. Sci., n.s., no. 247, vol. 62.
1918 Cytoplasmic inclusions. Part III. The spermatogenesis of
some other pulmonates. Ibid., n.s., no. 250, vol. 63.

Giatio-Tos AND GRANATA 1908 I mitocondri nelle cellule seminali maschili
di Pamphagus marmoratus (Burm). Biologica, vol. 2.

HouMGREN, N. 1902 Ueber den Bau der Hoden und die Spermatogenese von
Silpha carinata. Anat. Anz., Bd. 22.

McGreaor, J. H. 1899 The spermatogenesis of Amphiuma. Jour. Morph.,
supplement to vol. 15.

Meves, F. 1897 Ueber Struktur und Histogenese der Samenfaeden von Sala-
mandra maculosa. Arch. f. Mik. Anat., Bd. 50.

SHarrer, EH. L. 1917 Mitochondria and other cytoplasmic structures in the
spermatogenesis of Passalus cornutus. Biol. Bull., vol. 32.

Terni, T. 1914 Condriosomi, idiozoma e formazioni periidiozomiche nella
spermatogenesi degli anfibii (Ricerche sul Geotriton fuscus). Arch.
f. Zellforsch., Bd. 12.

Vespoysky, F. 1912 Zum Problem der Vererbungstraeger. Koenig. Boehm.
Gesellsch. d. Wiss., Prag.

Vornoy, D. 1916 Sur une formation juxta-nucléaire dans les éléments sexuels
du Gryllotalpa vulgaris, caduque & la fin de la spermiogenése. Compt.
Rend. Soe. Biol., T. 79. ;

EXPLANATION OF PLATES

All of the figures have been outlined as far as possible with the camera lucida,
those of plate 1 at an initial enlargement of approximately 2875 diameters, those
of plate 2 at approximately 3800 diameters. At so great enlargements it has,
of course, been necessary to correct the outlines extensively and to add much
of the finer detail free hand. In reproducing, the figures have been reduced
uniformly, those of plate 1 to an enlargement of 2300 diameters, those of plate 2
to 3350 diameters. In every case the method employed in the preparation of
the original object has been indicated.

ABBREVIATIONS

A, acrosome j, juxtanuclear body
a, acroblast n, nucleus
G, Golgi remnant s, spermatid remnant

PLATE 1
EXPLANATION OF FIGURES

Figures 1 to 19 are from Plethodon cinereus; figures 20 and 22 are from Dis-
sosteira carolina; the others are from Rhomaleum micropterum.

1 Late primary spermatocyte. (Flemming without acetic-hematoxylin. )

2 Metaphase first spermatocytedivision. (Cajal’s formol-uranium nitrate.)

3 Secondary spermatocyte. (Flemming without acetic-hematoxylin.)

4 Spermatid nucleus showing group of granules encircling the acroblast.
(Cajal-gold chloride-hematoxylin.)

5 Spermatid. Early formation of acrosome from acroblast. (Modified
Kopsch.)

6 Spermatid. Later stage in formation of acrosome. (Cajal-gold chloride—
hematoxylin.)

7 Spermatid. Final stage in formation of acrosome. (Cajal-gold chloride-
hematoxylin.)

8 Spermatid. Fixation of acrosome. (Champy-hematoxylin.)

9 Spermatid. Casting off of the acroblast (Golgi remnant). (Cajal.)

10 Spermatid. Showing proximal (?) centriole divided into two equal
parts. (Champy-hematoxylin.)

11 and 12 Spermatids. Progressive stages in the elongation of the nucleus.
Figure 12 shows only a portion of thenucleus. (Cajal-gold chloride-hematoxylin.)

13 and 14 Basal end of two adjacent spermatid heads in successive stages of
differentiation, to show separation and fusion of the components of the proximal
(?) centriole. (Champy-hematoxylin.)

15 Basal end of spermatid nucleus, to show continued separation of the
components of the proximal (?) centriole. (Champy-hematoxylin. )

16 to 18 Oblique sections through the basal end of the spermatid head, to
show possible origin of the membrane filament. (Champy-hematoxylin.)

19 Late stage in the differentiation of the spermatid. Elongation of the
ring centriole. (Champy-hematoxylin.)

20 Second maturation division. Late telophase. (Cajal.)

21 Cross-section through the nebenkern just prior to the complete disap-
pearance of thechromophilicsubstance. (Flemming without acetic-hematoxylin.)

22 Spermatid. Development of the central substance in the nebenkern.
(Cajal.)

23 and 24 Spermatids. Stages in the development of the acrosome. (Flem-
ming without acetic-hematoxylin. )

25 and 26 Spermatids. Later stages in the transformation of the acrosome.
(Flemming without acetic-hematoxylin.)

SPERMATOGENESIS IN AMPHIBIA AND INSECTS PLATE 1
ROBERT H. BOWEN

21

PLATE 2
EXPLANATION OF FIGURES

Figures 27 to 40 are from Lixus concavus; figures 41 to 46 are from Ceutho-
philus maculatus.

27 Spermatocyte (or late spermatogonium). (Modified Kopsch.)

28 to 30 Spermatids. Successive stages in the fusion of Golgi bodies to form
the acroblast. (Modified Kopsch.)

31 Spermatid. (Modified Kopsch.)

32 Cross-section of slightly elongated nebenkern. (Benda.)

33 Cross-sections of the nebenkern at approximately the stage of figure 34.
A, through the chromophilic substance; B, above (or below) the chromophilic
substance. (Benda.)

34 and 35 Spermatids, just prior to, and at the time of, disappearance of
the chromophilic substance. (Flemming-hematoxylin.)

36 to38 Late spermatids. Casting off of the acroblast. (Benda.)

39 Latespermatid. Golgi remnant passing backward along the tail. (Modi-
fied Kopsch.)

40 Spermatid, showing acrosome in final position. (Benda.)

41 Early spermatid. (Modified Kopsch.)

42 and 43. Spermatids. Development of the acrosome. (Flemming without
acetic-hematoxylin.)

44 Spermatid. Fixation of the acrosome. (Benda.)

45 Spermatid. Casting off of the acroblast. (Benda.)

46 Spermatid. Backward migration of the Golgi remnant and the juxta-
nuclear body. (Flemming without acetic-hematoxylin.)

22

SPERMATOGENESIS IN AMPHIBIA AND INSECTS PLATE 2
ROBERT H. BOWEN

23

Resumen por el autor, E. V. Cowdry

El material reticular como indicador de la reversi6n fisiol6gica
de la polaridad secretora de las células de la tiroides del
conejillo de indias

En las glindulas tiroideas de los conejillos de indias normal es
el material reticular no se encuentra invariablemente entre los
nuicleos y la cavidad de los foliculos, como se ha supuesto ge-
neralmente, sino que en algunos casos experimenta una activa
emigracién hacia el polo opuesto de la célula, la cual como de-
muestran otras pruebas, indica la existencia de una reversién
de la polaridad fisiol6gica mediante la cual la secrecién pasa
directamente a la circulacién en vez de acumularse primero en
los foliculos.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 27

THE RETICULAR MATERIAL AS AN INDICATOR OF
PHYSIOLOGIC REVERSAL IN SECRETORY POLARITY
IN THE THYROID CELLS OF THE GUINEA-PIG

E. V. COWDRY
Anatomical Laboratory of the Peking Union Medical College and the Laboratories
of the Rockefeller Institute for Medical Research, New York

TWO PLATES (FOURTEEN FIGURES)

It can easily be observed by the application of Da Fano’s
(20, p. 157) modification of Cajal’s silver-impregnation method
to the thyroid glands of guinea-pigs that the reticular material
is not always restricted to the zone of cytoplasm between the
nucleus and the follicular cavity, as has been supposed by all
previous workers (Negri, ’00, p. 61; ’00 a, p. 178; Holmgren, ’01,
p. 314, and Kolster, ’13, p. 128); but is found, in about one cell
in every five hundred, in the opposite pole near the peripheral
blood vessels. Detailed reviews of the literature relating to the
material are given by Duesberg (14) and Cajal (14).

A group of follicular cells is reproduced in figure 1 to illustrate
the average position assumed by the blackened reticular material
between the nucleus and the lumen. Reversal may take place
in single isolated cells, as shown at (A) in figure 2, or in large or
small groups of cells, as is illustrated in figures 3 and 4. On the
other hand, the reticular material may be found to be unusually
close to the lumen, as shown in figures 6 and 7. In very rare
cases, in which the epithelium is several layers in thickness, the
reticular material is extremely variable in position (fig. 8) and no
polarity can be distinguished.

In the acinus cells of the pancreas of the guinea-pig (fig. 9) it
is wholly different, because here the reticular material is, as far
as I can ascertain, invariably located between the nucleus and
the discharging pole of the cell, with strands sometimes extending
up on either side of the nucleus, and has been recorded in this

25

26 E. V. COWDRY

position by all investigators (Negri, ’00; v. Bergen, ’04, p. 533;
Bensley, ’11, p. 364; Kolster, 713; Cajal, 714, p. 169, and others).
The same generalization also holds for the salivary glands and,
to the best of my knowledge, for all glands in which the secretion
invariably passes in the same direction, at any rate under normal
conditions.

By means of a new method, Bensley (16, p. 48) has shown
that the thyroid is peculiar in another respect by confirming the
suspicion, which physiologists have for some time entertained,
that the secretion may change its direction and pass immediately
into the peripheral blood vessels without first being stored in
the follicular cavity. The technique consists of staining with
Brasilin in phosphotungstic acid after formalin-Zenker fixation
and of counterstaining with wasserblau in phosphomolybdic
acid. ‘This brings to light the true secretion antecedents in the
form of tiny vacuoles which contain a dilute solution similar in
its properties to the colloid of the follicular lumen, but less con-
centrated. Since the droplets always occur in the outer poles
of the cells, he concludes that the secretion ‘‘is destined to direct
transport into the vascular channels, and that the thyroid cell
presents a true reversal of polarity in accord with its endocrine
function.”’ He believes that only when ‘‘the rate of secretion is
in excess of body needs, the indirect mode of secretion comes in,
and the product of secretion is condensed and stored in the intra-
follicular cavity.”’ By way of further evidence, he calls to mind
the fact that in exocrine glands fat droplets ‘‘ practically always
make their appearance in the anti-secretory pole of the cell.”
In the thyroid they are confined to the end of the cell next to the
lumen, which would indicate that the secretion is discharged from
the opposite pole, remote from the lumen, in accordance with
his hypothesis. Still other evidence may be cited.

Norris’s (18, p. 462) suggestion that Bensley’s hypothesis of a
reversal is unnecessary because the interfollicular spaces may
represent the primitive lumina, so that the secretion has always
passed in this direction, is not substantiated. The presence of
the reticular material in so many cells between the nucleus and
the follicular lumen, as I have described it in the guinea-pig or

RETICULAR MATERIAL IN THYROID CELLS Da

as Cajal (14, p. 215) records it in other gland cells, between the
nucleus and the polo mundial, shows that the follicular cavities
are to be considered from the phylogenetic point of view as being
at one time in communication with the exterior (outside world).
If further proof is needed that these spaces represent the lumen
or duct of the ancestral gland, it is offered by the observation
(Cowdry, ’21), that the cells bordering them are uniformly
flagellated in the dogfish.

We have, then, a remarkable coincidence. Apparently in the
thyroid gland alone is there variability in the position of the
reticular material, and in the thyroid gland alone have we clear
evidence of physiologic reversal in the direction of secretion. In
my opinion, the two phenomena are related. Since the position
of the reticular material, like that of the centrosome, is a clue
to the polarity of the cell, preparations made by Da Fano’s
method will indicate the functional polarity, or, in other words,
the direction in which secretion is taking place at the moment
the preparations are made. This cannot be so safely inferred
from a consideration of the height of the epithelium and the
general appearance of the follicles as seen in routine hematoxylin
and eosin preparations. Neither is the amount of colloid to be
considered as a reliable clue, since it may have been produced
during a storage phase which preceded the examination of the
gland by a considerable interval of time.

The relatively small percentage of reversals in the position of
the reticular in the guinea-pigs which I have examined suggests
that the balance in production of secretion is in favor of storage
rather than of immediate discharge. Examination by Bensley’s
method, which is probably more delicate since it is also more
direct, would probably have failed to reveal any considerable
amount of secretion antecedent near the perifollicular net. In
the opossums, which he studied, in which the discharge into the
perifollicular spaces is probably more marked, I would predict
that the reticular material is often to be found between the
nucleus and the discharging pole. Judging from his figures, it
would appear that the nuclei have been forced toward the
lumen by the accumulation of secretion antecedents near the
periphery.

28 E. V. COWDRY

My contention regarding oscillation in secretion in the thyroid
receives some support from a study of the parathyroid glands in
which the reticular material has only thus far been recorded by
Kolmer (17, p. 272).!. He illustrates it in the form of a rather
dense and well-circumscribed network of a ring-like shape. Since
he finds that it is located at one pole of the nucleus (as in colum-
nar epithelia), he concludes that the cells possess two poles and
are cylindrical in shape, not roughly cubical as is generally sup-
posed to be the case, and, further, that they are disposed in rows
or end to end. My preparations of the parathyroid glands of
guinea-pigs reveal a blackened network which is usually some-
what more diffuse (figs. 10, 11, and 12). Quite frequently it is
cirecumnuclear in position, as is illustrated in one cell in figure 12.
But when the cells become grouped, the reticular material comes
to occupy a position between the nucleus and what may be con-
sidered to be the discharging pole. This condition is evident in
rare cases when the cells form follicle-like cavities, as shown in
particular by figure 138. The arrangement of cells in figure 12
is also suggestive. From this we may conclude that here also
the reticular material acts as an indicator of polarity assumed
under normal but unknown conditions.

Special interest attaches to the parathyroid glands because
their secretion is active, but unknown, and because true secretion
antecedents remain to be discovered within their cells. Perhaps
they may be more easily detected in these cells which assume a
certain measure of polarity, though it would be unsafe to say
that cells not definitely grouped are any lessactive physiologically.
The same amount of secretion antecedent in a polarized cell is
condensed into a relatively small area of the cytoplasm, whereas
in an unpolarized cell it is presumably spread throughout a much
larger extent of peripheral cytoplasm for transport in all direc-
tions. Just as in the thyroid, the first clues will probably be
found in hyperactive glands (if such can be produced). But we
have to remember that under normal conditions the secretion
antecedents may be present in such minute amounts or may be

1 Kolmer (’16, p. 507) also mentions the reticular material in the thyroid
gland, but does not describe its position within the cell.

RETICULAR MATERIAL IN THYROID CELLS 29

discharged so rapidly from the cells that they escape observation.
It is conceivable that the creation of passive congestion by ligat-
ing the venous drainage may cause a retention of secretion in
appreciable amounts.

With this idea of the reticular material as an indicator of secre-
tory polarity in mind, I think that a careful study of the hypo-
physis may indicate into which channels the secretion is actually
being discharged at the time the preparations are made. The
occurrence of this material in the cells of the anterior lobe has
already been recorded by Gemelli (’00) and by Tello (12). More
recently Addison (717, p. 448), attempting to confirm and extend
Gemelli’s work, has described a close-meshed reticulum which
is much larger in the basophiles than in the acidophiles. He is
so reticent regarding its nature that he prefers to call it the
“macula, until further information is forthcoming.” My pre-
liminary studies with the guinea-pig have failed to establish any-
thing approaching an orderly arrangement (fig. 14). In order
to obtain decisive results, the silver preparations will have to be
combined with vascular injections, and will have to be recon-
structed in serial sections, because the relations of the cells are
so intricate.

Cajal (14, p. 214) devotes several pages to a general considera-
tion of the phenomena of polarization of the Golgi apparatus (or
reticular material), mentioning particularly cases of ontogenetic
reversal in its position in nerve cells. It is interesting to note
also that Tello’s (13, p. 145) investigations on mammary-gland
carcinomata bring to light a progressive depolarization in its posi-
tion as the tissue assumes the characteristics of a neoplasm.

Two interesting cases of experimental reversal are described in
the stomach and in the kidney which show the sensitivity of the
reversal mechanism.

D’ Agata (10, p. 518) found that the position of the reticular
material in the gastric cells is reversed sixteen hours after oper-
ating on the stomach of triton. His figures show clearly that the
material has taken up a position between the nucleus and the
proximal border of the cell; that is to say, it is now in the pole of
the cell which is generally considered to be the antisecretory. If

30 E. V. COWDRY

our hypothesis is correct, this reversal would mean the temporary
passage of material from the lumen in the direction of the per-
ipheral blood vessels. Absorption from the lumen, which nor-
mally takes place to some extent, is probably accelerated as a
result of the operation. We would not expect to be able to
induce so simply experimental reversal in the position of the
reticular material in the acinus cells of the pancreas or in the
salivary glands, because absorption, with the accompanying
tendency to the production of a current in an unusual direction,
is in them at a minimum.

Basile (714, p. 3) discovered that unilateral nephrectomy pro-
duces definite changes, possibly compensatory in nature, in the
reticular material of the remaining kidney. From its normal
position between the nucleus and the lumen it migrates, in both
the tubuli contorti and recti, to a new position between the nu-
cleus and the base of the cell. This would, in my judgment, indi-
cate the predominance of absorption over excretion and would
seem to pave the way for a study of the physiology of the kidney
from a new angle.

But we are laboring more or less blindly because so little is
known regarding the true nature of the reticular material. The
chief difficulty is that we have thus far been unable to study it
directly in living cells. It is a simple matter to give a long list
of chemicals which destroy it during fixation, and to enumerate
stains, like. resorecin-fuchsin, which have been known in rare
instances to color it, without mentioning any positive reaction of
value. By virtue of its affinity for silver (methods of Golgi and
Cajal) and of the fact that it blackens after prolonged treatment
with osmic acid (method of Kopsch), it may be studied closely
in fixed tissues. Though both of these methods are more or less
unsatisfactory, the results which they yield are always mutually
confirmatory; so that one is justified in concluding that by their
characteristic morphology and location, the blackened networks
reveal the existence of some unknown and hitherto unrecognized
material in the living cell. This conclusion is supported by the
further observation that fixation in several fluids, such as Zenker,
under certain conditions, and a mixture of formalin, potassium

RETICULAR MATERIAL IN THYROID CELLS ol

bichromate, and mercuric chloride, as reeommended by Bensley
(10, p. 192), brings to hight, in most tissues, a system of clear
eanals which bear a very close resemblance in shape and posi-
tion to the blackened networks (compare figs. 5 and 1); thus there
are in reality three methods, two positive and one negative,
which yield the same results.

We infer that this unknown reticular material is of considerable
physiologic importance in the living cell; because our methods
show that the blackened networks (or clear canals) are very
widely distributed in animal cells and that, in some cases, they
undergo characteristic and progressive alterations during develop-
ment and in different physiologic and pathologic conditions.

We also infer that the reticular material is fluid in consistency
because there is no indication of the existence of a rigid frame-
work-in follicular cells teased out in salt solution, and the dis-
tribution andmovement of cytoplasmic granules, like mitochondria
do not seem to be restricted. Centrifuging fragments of thyroid
for upwards of forty-five minutes at 3,500 revolutions per minute
does not displace it, so that its specific gravity cannot differ
greatly from the remainder of the cytoplasm. Further evidence
regarding its fluidity I have presented elsewhere (’21, p. 7).

It has been suggested that in other glands, like the pancreas,
the reticular material plays a definite part in secretion. Saguchi
(20, p. 393) is willing to go so far as to say that ‘“‘the intracellular
network consists of secreting material which is to be extruded
either directly into the lumen or indirectly into the intercellular
eapillary.”’ This is difficult to deny in the thyroid or elsewhere
because the entire cytoplasm, to say nothing of the nucleus, is
either directly or indirectly involved. If our inference regarding
its fluidity iscorrect, many chemical reactions of diverse character
may take place in it and along its changing surfaces. It is prob-
ably quite heterogeneous in composition and one of the most
active areas of the cytoplasm.

THE AMERICAN JOURNAL OF ANATOMY, VOL, 30, NO. 1

we)
bo

E. V. COWDRY

SUMMARY

In the thyroid glands of normal guinea-pigs the reticular
material is not invariably found between the nuclei and the
follicular lumen, as is generally supposed, but in some cases under-
goes an active migration to the opposite pole of the cell, which,
together with other evidence at hand, indicates the existence of
a reversal in physiologic polarity whereby the secretion is dis-
charged directly into the circulation, instead of being first stored
within the follicles.

BIBLIOGRAPHY

Appison, W. H. F. 1917 Cell changes in the hypophysis of the albino rat
after castration. Jour. Comp. Neur., vol. 28, pp. 441-463.

Basin, G. 1914 Sulle modificazioni dell’apparato reticolare interno di Golgi
nell’ epitelio renale di animali refrectomizzati. Internat. Montschr.
f. Anat. u. Physiol., Bd. 31, 8. 1-9.

Benstey, R. R. 1910 On the nature of the canalicular apparatus of animal
cells. Biol. Bull., vol. 19, pp. 179-194.
1911 Studies on the pancreas of the guinea pig. Am. Jour. Anat.,
vol. 12, pp. 297-3888.
1916 The normal mode of secretion in the thyroid gland. Am. Jour.
Anat., vol. 19, pp. 87-54.

v. Beraen, F. 1904 Zur Kenntnis gewisser Strukturbilder (Netzapparate,
Saftkindlehen, Trophospongien) im Protoplasma vershiedener Zel-
arten. Arch. f.mikr. Anat., Bd. 64, S. 498-574.

Casau, S. R. 1914 Algunas variaciones fisiologicas y patolégicas del aparato
reticular del Golgi. Trab. Lab. Invest. Biol., Madrid, vol. 12, pp.
127-228.

Cowpry, E. V. 1921 The reticular material of developing blood cells. Jour.
Exper. Med., vol. 33, pp. 1-11.
1921 Flagellated thyroid cells in the dog-fish (Mustelus canis).
Anat. Rec.

D’Aaata, G. 1910 Sulle modifieazioni dell’ apparato reticolare interno nell’
epitelia della mucosa gastrica. Bull. d. Soe. med.-chir. di Pavia,
vol. 23, pp. 519-522.

Doursspera, J. 1914 Trophospongien und Golgischer Binnenapparat. Anat.
Anz., Bd. 46, S. 11-80.

Fano, C. Da. 1920 Method for the demonstration of the Golgi apparatus in
nervous and other tissues. Jour. Roy. Mier. Soc., no. 211, pp. 157-161.

Gemewur, EH. 1900 Recherche sperimentali sulla struttura della ghiandola
pituitaria nei Mammiferi. Boll. Soe. Med.-Chir. di Pavia, vol. 14,
pp. 231-242.

Houtmeren, Emit 1901 Neue Beitriige zur Morphologie der Zelle. Ergebn.
d. Anat. u. Entwceklngsgesch., Bd. 11, 8. 274-329.

RETICULAR MATERIAL IN THYROID CELLS 30

Kotmer, Wauter 1916 Uber einige durch Ramon y Cajal’s Uran-Silber-
methode darstellbare Strukturen und deren Bedeutung. Anat. Anz.,
Bd. 48, 8. 506-519.

1917 Zur Histologie der Parathyroidea und Thyroidea. Ibid., Bd. 50,
S. 271-277.

Kouster, R. 1913 Ueber die durch Golgi’s Arseniek—und Cajal’s urannitrat—
Silbermethode darstellbaren Zellstruckturen. Verhandl. d. anat.
Gesellsch., Bd. 23, S. 124-132.

Neari, A. 1900 Di una fina particolarita di struttura delle cellule di aleune
ghiandole dei mammiferi. Boll. d. Soc. med.-chir. di Pavia, vol.14,
pp. 61-70.

1900 a Ueber die feinere Struktur der Zellen mancher Driisen bei
den Saiugetieren. Verhandl. d. anat. Gesellsch., Bd. 14, S. 178-181.

Norris, E. H. 1918 The early morphogenesis of the human thyroid gland.
Am. Jour. Anat., vol. 24, pp. 443-466.

Saqgucut, 8S. 1920 Studies on the glandular cells of the frog’s pancreas. Am.
Jour. Anat., vol. 26, pp. 347-421.

Tewtio, F. 1912 Communicacion 4 la Sociedad Espafiola de Biologia (quoted
from Duesberg).

1913 El reticulo de Golgi en las células de algunos tumores y en las
del granuloma experimental peroducido por el Kieselgur. Trab. Lab.
Invest. Biol., Madrid, vol. 11, pp. 145-161.

DESCRIPTION OF FIGURES

All the drawings have been made with a1.5-mm. Zeiss apochromatie objective,
no. 10 Spencer ocular and camera lucida, and are reproduced without reduction.

PLATE 1
EXPLANATION OF FIGURES

1 Group of follicular cells showing the usual form and position of the reticu-
lar material from a preparation made by Cajal’s uranium and silver nitrate
method, which blackens it.

2 Follicular cells prepared by Da Fano’s cobalt nitrate silver method. At
(A) one large cell presents an apparent reversal in polarity, since the blackened
reticular material is on the opposite side of the nucleus remote from the follicular
cavity.

3 <A row of cells in all of which the position of the reticular material is re-
versed, from a preparation made by the same method.

4 Follicular cells showing different degrees of reversal in position as one
passes from left to right, from another guinea-pig treated by the same method.

5 Follicular cells fixed in Zenker’s fluid and stained with iron hematoxylin,
showing a system of clear canals in the same position as the blackened material
illustrated in figure 1.

6 Cells prepared by Da Fano’s cobalt nitrate silver method, showing reticu-
lar material which has moved up unusually close to the follicular cavity and which
exhibits vesicle-like enlargements. Compare this with its average position and
form illustrated in figure 1.

7 The same, without the swellings.

8 Three layers of epithelial cells bordering the follicular lumen and showing
no sign of polarity as indicated by the variable location of the blackened reticular
material, same method.

PLATE 1

RETICULAR MATERIAL IN THYROID CELLS

E. V. COWDRY

PLATE 2
EXPLANATION OF FIGURES

9 A pancreatic acinus showing perfectly definite secretory polarity with the
blackened reticular material placed in each cell between the nucleus and the
lumen, same method.

10 Group of parathyroid cells from a piece of tissue centrifuged in the direc-
tion of the arrow in normal saline for forty-five minutes, at about 4,500 revolu-
tions per minute, same method.

11 Parathyroid cells fixed in Bensley’s acetic-osmic-bichromate mixture
and impregnated with 2 per cent osmic acid according to the method of Kolatchev
as suggested by Ono. The reticular material is blackened, but is not very defi-
nitely oriented.

12 Parathyroid cells prepared by Da Fano’s cobalt nitrate silver method in
which the blackened reticular material shows but slight evidence of polar
arrangement.

13 Cluster of parathyroid cells from the same centrifuged tissue as figure 10,
showing a grouping of the cells with definite orientation of the reticular material
between the nucleus and the lumen.

14 A small group of cells from the anterior lobe of the hypophysis, also
prepared by Da Fano’s method, and showing how variable is the position of the
reticular material in the cytoplasm. There appears to be no conformity in its
location even in adjacent cells.

36

PLATE 2

RETICULAR MATERIAL IN THYROID CELLS

E, V. COWDRY

Resumen por el autor, Warren H. Lewis
El endotelio en cultivo de tejidos

Los cultivos fueron hechos del modo corriente utilizando el
medio Locke-Lewis. El higado del embrién de pollo de siete
dias consiste de higado y células endoteliales; cada uno de estos
dos tipos emigra hacia el cubreobjetos de una manera caract-
eristica. El] endotelio forma un reticulo laxo de células alar-
gadas més o menos adherentes entre si por medio de sus
extremos y procesos. El caracter de este reticulo varia con-
siderablemente, pero los procesos celulares no son tan abundantes
como en las células mesenquimatosas multipolares. En la
periferia, y también en una forma de degeneracion, las células
pueden aislarse completamente unas de otras. Estos hechos
indican que el reticulo es adherente en vezde ser sincicial.

En los cultivos j6venes las mitocondrias aparecen principal-
mente en forma de filamentos con un ntimero variable de bas-
tones y grdinulos. A medida que envejecen dichos cultivos las
mitocondrias tienden a disponerse radialmente a partir del cen-
triolo y centrosfera en vias de crecimiento. También tienden a
fragmentarse en bastones y granulos. Su cardcter y cantidad
varian considerablemente. Grdanulos y vacuolas que presentan
una mareada afinidad con el rojo neutro (granulos de degenera-
cion) se acumulan gradualmente en las células y presentan una
tendencia a amontonarse alrededor del centriolo y centrosfera
dilatada. Las células binucleadas son comunes. En los cul-
tivos mids viejos, en los cuales los cambios degenerativos son evi-
dentes, los nticleos a menudo se hacen irregulares y Ilenos de
dentellones, dividiéndose en varios nuicleos mas pequenos El
ectoplasma a veces presenta fibrillas longitudinales semejantes
a las de las fibras musculares lisas depués de la fijacién. Di-
chas fibrillas no pueden distinguirse en la célula viva.

Translation by José IF’. Nonidez
Cornell Medical College, New York

AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 27

ENDOTHELIUM IN TISSUE CULTURES

WARREN H. LEWIS
Carnegie Laboratory of Embryology, Johns Hopkins Medical School

FIVE PLATES (TWENTY-FOUR FIGURES)

INTRODUCTION

It is but natural that investigators in the field of tissue cul-
ture should have had and should still have more or less difficulty
in identifying the various types of cells that grow out from ex-
plants of embryonic tissues. Most explants contain several
types of cells that migrate out into the medium, some of which
are comparatively easy to identify.

Nerve fibers from the central nervous system have been ob-
served by Harrison (’07, ’10), Ingebritsen (13), and Levi (’16 a),
and those from the sympathetic system by Lewis and Lewis
(12a), and Matsumoto (’20). They are always quite charac-
teristic. Endodermal membranes from the cells lining the ali-
mentary tract and allantois were not at first identified as such
(Lewis and Lewis, 711; Lambert, ’12), but later their origin was
established (Lewis and Lewis, ’12b) and their recognition is
now simple. Ectoderm from the skin (Lambert, 712) and the
amnion (Lewis and Lewis, 712 b) grows out in the form of a
membrane or sheet, as does also the pigmented epithelium from
the retina (Luna, ’17, fig. 1; Smith, ’20). In the frog, likewise,
the epithelial cells grow out as membranes (Uhlenhuth, 714,
"15; Matsomoto, 718). Liver cells form membranes similar
to the endoderm and are easily identified (Lynch, ’21). The thy-
roid-gland cells grow out either as tubules or membranes (Car-
rel and Burrows, ’11 a,’11b). Renal epithelium from the tubules
likewise-grows out in the form of membranes or sheets (Lewis
and Lewis, 712 b) and sometimes as tubules (Reinhoff!). Blood-

: Unpublished.

39

40) WARREN H. LEWIS

cells and wandering cells from the spleen (Foot, 712), bone-
marrow (Erdmann, *17), lymph nodes (Lewis and Webster,
21a, ’21b), and thymus (Pappenheimer, 713) seem always to
migrate out as isolated cells and to remain so. Skeletal muscle
has been observed by M. R. Lewis (15, ’19), and Lewis and Lewis
(17 a), and premuscle by Congdon (715). This tissue is usually
easy to recognize by its elongated multinuclear fibers and pecu-
liar cytoplasm. Heart muscle has been studied by Burrows
(10, ’12), Congdon (15), and more especially by Levi (’16b,
19). Some of the figures of heart muscle shown by the last-
named investigator correspond to our own unpublished ones,
but this is not the case with those of Burrows and of Congdon,
except possibly Congdon’s figure 5. Smooth muscle resembles
more the ordinary mesenchyme than either skeletal muscle or
heart muscle and has been observed by Lewis and Lewis (717 b)
and by M. R. Lewis (’20 a).

We have been using in a rather loose fashion the terms mes-
enchyme, connective tissue, and fibroblast to designate the reticu-
lar radiating outgrowths in tissue cultures, because we were
not sure of the exact identity of the tissue. I think most in-
vestigators working with tissue cultures have felt, as we do, that
in using these terms they were probably dealing with several
types of cells, namely, mesenchyme (including reticulum), fibro-
blasts, and other types of fixed connective-tissue cells, endothe-
lium, and mesothelium. The literature is full of figures of these
mesenchymal tissues, and among them are some which are prob-
ably endothelium. Most explants contain capillaries with
endothelial cells and in many cultures these cells probably mi-
grate out, although they may not be recognized as endothelium.

The present study is devoted to a consideration of the endo-
thelial cells that migrate out from the explants of embryonic
chick liver. It is well known that the liver, after ninety-six
hours of incubation contains but two types of cells, the liver cells
proper and the endothelium of the sinusoids (Minot, ’00). It
seemed to offer, therefore, an excellent opportunity for the study
of endothelial outgrowths, as no difficulty should be encoun-
tered in distinguishing the liver-cell membrane from the loose

ENDOTHELIUM IN TISSUE CULTURES 41

reticular outgrowths (Lynch, ’21). I have regarded these retic-
ular outgrowths as endothelium. They differ in character
from the reticular outgrowths of ordinary subcutaneous mesen-
chyme, as can be seen by comparing figures | and 2 of subcutane-
ous mesenchyme with figures 3 to 12 of endothelium.

MATERIAL AND METHODS

The explants were all from the livers of chick embryos of five
to ten days’ incubation. The cultures were made in the usual
manner with Locke’s solution (NaCl, 0.9 gram; CaCh, 0.024
gram; KCl, 0.042 gram; NaHCO;, 0.02 gram; dextrose, 0.5
gram, H.O, 100 ec.), 80 ec., plus chicken bouillon, 20 ce. The
chicken bouillon was made according to the usual bacteriological
method, except that freshly killed chicken was used in the place
of beef. The hydrogen-ion concentration of the solutions varied
from pH 6.4 to pH 7.6, but was usually about pH 6.8 to pH 7.2.

The figures are all from untouched photographs. The cul-
tures from which they were taken were first subjected to a weak
solution of janus green or neutral red, or the two in combination,
until the mitochondria or the granules (and vacuoles) or both
were stained. The cultures were than fixed with iodine vapor
by placing a small flake of pure iodine on the bottom of the
hollow-ground slide. The iodine vapor rapidly tinted the cells
a beautiful yellow-brown. The mitochondria when stained with
janus green became a very dark brown (almost black) and the
neutral red granules became a dark red-brown. ‘These colors
are very advantageous for photography.

GENERAL CHARACTERISTICS

The endothelial cells migrate out from the liver explants onto
the cover-glass in the form of a loose reticulum of elongated
cells that are more or less adherent to one another by their ex-
tremities and processes. ‘The character of this reticulum varies
considerably in different cultures and in different regions of the
same culture, as will be seen by comparing figures 3 to 12. Near
the explant and in the middle of the outgrowth the cells are
elongated and slender, while at the periphery, where the crowding

42 WARREN H. LEWIS

is less, they tend to flatten out in various directions. ‘This is
often still more marked in the isolated cells near the periphery
The explanatioy of this is probably not in a longitudinal polarity
that disappears as the cells move peripheralward, but in the
interplay of surface tension and of the differential adhesiveness
of the cells for each other and for the cover-glass. The cells
have apparently a greater adhesiveness for the cover-glass
than for each other, and at the periphery, where they have more
space, they are drawn out more and more on the cover-glass
by the tension pull of the surface film of the fluid medium which
lies against the cover-glass, until a balance between capillary
attraction and cohesion is reached. ‘Tait (18) has recently de-
scribed the spreading-out of certain blood-cells on glass as due to
capillary attraction or, in other words, to surface-tension phe-
nomena. The endothelial cells are evidently sticky and semifluid
and are subject to the same physical forces as the blood-cells
described by Tait. There may also be alterations, both local
and general, in the consistency of the cytoplasm, which would
favor the more extensive spreading-out of the cell. The consis-
tency or viscosity of protoplasm is probably a variable char-
acter, as shown by Chambers (717) and Seifriz (20). The mi-
gration of the cells probably depends on alternating changes
in the consistency of the protoplasm with changes in surface
tension, as suggested by Loeb (20, ’21) for the amoeboid move-
ments of the blood-cells of Limulus.

In the many cultures examined we have never seen spread-out
endothelial cells from the liver with long, slender, fine-branched
processes such as are shown in figures 1 and 2 from subcutaneous
tissue. Figures 7 and 8 show the slender more elongated type
of cell, and figures 3, 4, 9, and 11, the more common type, which
possesses a broad, thin, lateral expansion. Figures 5 and 6,
a more unusual variety, show the cells forming a somewhat
membranous or sheet-like outgrowth resembling the mesothe-
lium-like membranes in cultures from the heart and intestine.
They are different, however, and mesothelial membranes have
not been observed in these liver cultures.

ENDOTHELIUM IN TISSUE CULTURES 43

The contrast between the endothelial cells and the liver cells
is always quite marked, as shown in figures 5 and 12. Some-
times the endothelial cells have exceedingly long and slender
processes which extend out from the ends of the cells parallel
to each other and to the long axes of the cells.

It is very difficult to determine whether the reticulum formed
by the endothelial cells is a syneytium or not. In most places
it is impossible to decide between actual fusion and mere adhesion.
Occasionally it is clearly evident that the connections are only
adhesions and that each cell preserves its individuality. This
together with the fact that cells at the periphery readily become
isolated from one another, would lead one to believe that actual
continuity does not exist at any time. Again, under certain
conditions, cells retract their processes and tend to round up,
losing all connection with neighboring cells; this also would
indicate adhesion and not fusion. The difficulty of determining
the exact status of the exceedingly thin processes that extend
out onto neighboring cells, especially where the cells are closely
packed together, has led us in the past to consider that we were
dealing with a syncytium, but we are inclined now to doubt this,
both for endothelium and mesenchymal reticuli in general.

CELL STRUCTURES

Mitochondria. In young cultures the mitochondria are mostly
in the form of threads of varying lengths and forms and often
with no definite orientation. In addition there are often rods
and granules (fig. 13). Sometimes, even on the first day, they
may show a more or less radial arrangement about the centriole
region. The conditions of the mitochondria in different cultures
of the same age, and even in different cells of the same culture,
vary so tremendously that it is difficult to give a general descrip-
tion of their appearance (figs. 18 to 20). No two cells are ever
exactly alike. As the culture ages the mitochondria tend to
become more or less radially arranged about the centriole and
enlarging centrosphere (figs. 14, 15) and the threads tend to
break up into rods and granules until finally the cells contain
a large (giant) centrosphere and mitochondrial granules (figs.

44 WARREN H. LEWIS

16, 19). ‘The process is essentially the same as that already
shown for fibroblasts and mesenchymal cells (W. H. Lewis,
19, 720).

I have before me seventy photographs (at 1450 diameters) of
endothelial cells from this series, which were taken especially for
the mitochondria, and it is difficult to select therefrom a few
that will adequately convey to the reader an idea of the great
variability displayed by these cells. In some cultures varying
numbers of cells, as early as the third day, contained only granu-
lar mitochondria. ‘This condition, however, may be delayed;
some cells, even as late as the tenth day of cultivation, still
showed thread-like, as well as granular and rod-shaped, mito-
chondria. Most of the cells, even up to the tenth day, contained
all three types, granules, rods, and threads, varying in length
thickness, and form. Variations in the amount of mitochondrial
substance were sometimes quite marked. Presumably the mito-
chondrial complex is much more constant under the normal
conditions within the embryo, and the great variations in the
cultures are to be attributed to the variations in the abnormal
conditions of the cultures. They do not appear to be due to
differences in hydrogen-ion concentration from pH 6.4 to pH 7.6,
nor to the age of the embryo, since great variations may occur in
two successive cultures of the same series or even in the same
culture. The most probable factor seems to be in the thickness
of the medium drop and the accompanying evaporation in the
air chamber, with consequent variations in the oxygen supply
to the cells, dependent on the thickness of the fluid separating
them from the air in the chamber of the hollow slide.

Granules and vacuoles. Granules and vacuoles, which have
a marked affinity for neutral red, methylene blue, and brilliant
cresyl blue, gradually accumulate in these cells and vary as
much as they do in the fibroblasts and mesenchyme cells (Lewis
and Lewis, 715; W. H. Lewis, ’19, ’20). The rate at which they
make their appearance varies in different cultures; as do like-
wise the relative proportions and the size of the granules and
vacuoles (figs. 21 to 23). We are still of the opinion that these
granules and vacuoles bear a relation to degenerative changes

ENDOTHELIUM IN TISSUE CULTURES 45

going on within the cells (W. H. Lewis, 719). Recent work has
tended to confirm this view. Mrs. Lewis (’20 b, ’20 c) has shown
that the addition of bacillus typhosus to the cultures causes a
very rapid formation of similar vacuoles, and still more recently
she has found that with the absence of dextrose from culture
media vacuolization of the cells is more rapid and extensive than
when dextrose is added. Miss Prigosen (’20, ’21), working in
our laboratory, found that the cells in film preparations of the
subcutaneous tissue of chick embryos showed a rapid accumu-
lation of granules and vacuoles that had a great affinity for
neutral red.

Centriole and centrosphere. The centriole was not observed
in the living cells, but in the fixed material could often be rec-
ognized near the nucleus as a small granule about which the
mitochondria and neutral red granules tended to accumulate,
the former often assuming a more or less radial arrangement.
In older cultures there frequently develops about the centriole
a centrosphere which gradually enlarges (figs. 14, 15, 16; 19;
20,21). This area varies in character ;1t may be quite homogene-
ous, as in figures 16, 19, 20, or granular, as in figures, 14, 15.
The enlargement of the centrosphere has not been followed in
detail in these cells, but the process seems to be similar to that
previously described for the mesenchyme cells (W. H. Lewis,
95220).

Nucleus. The nuclei are oval in form, long and narrow in the
elongated cells, and short and plump in the spread-out flattened
cells. The contour is usually smooth, but not infrequently in
the older cultures it is uneven, being more or less irregular and
indented. This appears to be the first indication of the process
of budding and amitosis which results in the splitting of the
nucleus into several, sometimes four or more, smaller nuclei
(fig. 23). This process seems to take place only in the older
cultures where other degenerative changes are evident. When
a culture shows such changes there are usually many cells thus
affected exhibiting various stages of the process. I have not
actually followed this amitotic division in the living cells, but the
evidence from the fixed material is very conclusive. In addition

46 WARREN H. LEWIS

to this degenerative fragmentation of the nuclei, more normal
binucleate cells are not uncommon in cultures of all ages.

Binucleate cells. Macklin (16) found that binucleate cells
were more numerous in cultures from the hearts of chick embryos
of five days’ than in those of eight days’ incubation and that
they were more numerous in the two-day- than in the one-day-
old cultures. He found, furthermore, that the average binu-
cleate cell was approximately twice as large as the mononucleate,
and that each nucleus was very similar in size, shape, and general
appearance to the nuclei of the mononucleate cell—findings which
my own observations have confirmed. I have further noted
that there is a very decided increase in the mitochondrial con-
tent. Figure 10 shows a very large binucleate cell—a giant
compared to its neighbors. Each nucleus seems to be as large
as normal, while the cytoplasm is more than twice that of the
largest mononuclear. ‘The mitochondria are of the same general
character (granules and rods) as those in the neighboring cells,
but are almost, if not fully, fourfold in number.

Nucleolus. The nucleoli vary in number from one to four
and also vary in position, size, and form. In some of the older
cultures the nucleoli are extruded from the nucleus, usually
from one end (fig. 24). The exact details of this process have
not as yet been carefully followed. The nucleolus in some way
reaches the end of the nucleus and les against its membrane.
Disintegration then takes place in this region, the nuclear mem-
brane disappears, a vacuole-like area develops in the adjoining
cytoplasm, and the nucleolus eventually comes to le within it.

Cytoplasm. In the living cultures both ectoplasm and endo-
plasm appeared homogeneous. In the fixed material the ecto-
plasm was sometimes homogeneous and sometimes striated
(figs. 16 and 17). ‘These striae were not seen in the living cells,
but formed as the cytoplasm coagulated under the influence of
the iodine vapors. Ido not believe they correspond to preformed
structures. They varied in size and ran out into processes
often to their very tips. This would seem to indicate that the
peculiarity of the ectoplasm which causes it to coagulate into
fibrillae is a molecular thing associated with tension, due to the

ENDOTHELIUM IN TISSUE CULTURES 47

surface tension pull. This striation was similar to, but not so
marked as that seen in the smooth-muscle cells in cultures, and
it suggests that there is an unusual amount of contractile sub-
stance in endothelium which is interesting in connection with
recent physiological work on the contraction of capillaries by
Dale, Krogh, and Hooker.

The endoplasm became very finely granular on coagulation
and in it were embedded most of the mitochondria and granules.

BIBLIOGRAPHY

Burrows, M. T. 1910 The cultivation of tissues of the chick embryo outside
the body. J. Am.M. Ass., vol. 55.
1911 The growth of tissues of the chick embryo outside the animal
body, with special reference to the nervous system. Jour. Exp.
Zool., vol. 10.
1912 Rhythmische Kontraktionen der isolierten Herzmuskelzelle
ausserhalb des Organismus. Miinchen. med. Wochnschr., No. 27.

CarRREL, A., aNnDM.T. Burrows 1911 a Cultivation of tissues in vitro and its
technique. J. Exper. Med., vol. 13.
1911 b Cultivation in vitro of thyroid gland. J. Exper. Med., vol. 13.

CHAMBERS, R. 1917 Microdissection studies. II. The cell aster: a reversible
gelation phenomenon. Jour. Exp. Zodl., vol. 23, pp. 483-504.

Conapon, E.D. 1915 The identification of tissues in artificial cultures. Anat.
Rec., vol. 9.

ErpMANN, Roopa 1917 Cytological observations on the behavior of chicken
bone-marrow in plasma medium. Am. Jour. Anat., vol. 22.

Foot, N. 1912 Ueberdas Wachstum von Knochenmark in vitro, etc. Beitr. z.
path. Anat., Bd. 53.

Harrison, R. G. 1907 Observations on the living developing nerve fiber.
Anat. Rec., vol. 1.
1910 The outgrowth of the nerve fiber as a mode of protoplasmic
movement. Jour. Exp. Zodl., vol. 9.

INGEBRITSEN, R. 1913 Studies on the degeneration and regeneration of axis
cylinders in vitro. J. Exper. Med., vol. 17.

LamsBertT, R. A. 1912 Variations in the character of growth in tissue cultures.
Anat. Rec., vol. 6.

Levi, G. 1916a Sull’ origine delle reti nervose nelle colture ditessuti. Rend.
d. R. Accademia dei Lincei, vol. 25.
1916 b Migrazione di elementi specifici differenziati in colture di
miocardio e di muscoli scheletrici. Arch. per le Sci. Med., vol. 40.
1917 Connessioni e struttura degli elementi nervosi R. Accademia
dei Lincei. '
1919 Nuovi studi su cellule coltivate ‘in vitro.’ Arch. ital. di Anat.
e di Emb., vol. 16.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. L

48 WARREN H. LEWIS

Lewis AND Lewis 1911 The cultivation of tissues from chick embryos in
solutions of NaCl, CaClz, KCl, and NaHCO 3. Anat. Rece., vol. 5.
1912a The cultivation of sympathetic nerves from the intestine of
chick embryos in saline solutions. Anat. Rec., vol. 6.

1912 b Membrane formations from tissues transplanted into artificial
media. Anat. Rec., vol. 6.

1915 Mitochondria (and other cytoplasmic structures) in tissue
cultures. Am. Jour. Anat., vol. 17.

Lewis, M. R. 1915 Rhythmical contraction of the skeletal muscle tissue
observed in tissue cultures. Am. J. Physiol., vol. 38.

1916 Sea water as a medium for tissue cultures. Anat. Rec., vol. 10.

Lewis AND Lewis 1917 a Behavior of cross-striated muscle in tissue cultures.
Am. Jour. Anat., vol. 22.

1917 b The contraction of smooth muscle cells in tissue cultures.
Am. J. Physiol., vol. 44.

Lewis, W. H. 1919 Degeneration granules and vacuoles in the fibroblasts of
chick embryos cultivated in vitro. Johns Hopkins Hosp. Bull.,
vol. 30.

Lewis, W. H. 1920 Giant centrospheres in degenerating mesenchyme cells of
tissue cultures. J. Exper. Med., vol. 31.

Lewis, M.R. 1920 Muscular contraction in tissue cultures. Contributions to
Embryology, vol. 9. Carnegie Inst. of Wash., Pub. 272.

1920 a The rapid formation of vacuoles due to the presence of bacillus
typhosus in the cells of tissue cultures. Anat. Rec., vol. 18.

1920 b The formation of vacuoles due to bacillus typhosus in the
cells of tissue cultures of the intestine of the chick embryo. J. Exper.
Med., vol. 31.

1921 The formation of vacuoles in the cells of tissue cultures owing
to the lack of dextrose in the media. Anat. Rec., vol. 20.

Lewis, W.H., ano L. T. Wesster 1921 a Migration of lymphocytes in plasma
cultures of human lymph nodes. J. Exper. Med., vol. 33.

1921 b Giant cells in cultures from human lymph nodes. J. Exper.
Med., vol. 33.

Lors, Leo 1920 The movements of the amoebocytes and the experimental
production of amoebocyte (cell-fibrin) tissue. Washington Univ.
Studies, vol. 8; Scientific Series, no. 1, pp. 38-79.

1921 Amoeboid movement, tissue formation and the consistency
of protoplasm. Science, N.S., vol. 53, p. 261.

Luna, EK. 1917 Note citologiche sull’ epitelio pigmentato della retina coltivato
“in vitro.’’ Arch. ital. di Anat. e di Emb., vol. 15.

Lyneu, R. 8. 1921 The cultivation in vitro of liver cells from the chick
embryo. Amer. Jour. Anat., Vol. 29, p. 281.

Mackuin, C. C. 1916 Binucleate cells in tissue cultures. Contributions to
Embryology, Carnegie Inst. of Wash., Pub. 224.

Matsumoto, 8. 1918 Contribution to the study of epithelial movement. The
corneal epithelium of the frog in tissue culture. Jour. Exp. Zodl.,
vol. 26.

ENDOTHELIUM IN TISSUE CULTURES 49

Marsumoto, T. 1920 The granules, vacuoles and mitochondria in the sympa-
thetic nerve fibers cultivated in vitro. Johns Hopkins Hosp. Bull.,
vol. 31.

Minor, C. 8. 1900 Ona hitherto unrecognized form of blood circulation with-
out capillaries in the organs of vertebrata. Proc. Boston Soc. Nat.
Hist., vol. 29.

PAPPENHEIMER, A. M. 1913 Further studies of the histology of the thymus.

Am. Jour. Anat., vol. 14.

PricosEN, R. E. 1920 Vacuoles formed in certain cells studied under ab-
normal conditions. Anat. Rec., vol. 18.
1921 The formation of vacuoles in connective tissue cells due to
abnormal conditions. Johns Hopkins Hosp. Bull., vol. 32.

Serrriz, W. 1920 Viscosity values of protoplasm as determined by micro-
dissection. Bot. Gazette, vol. 70.

SmitH, D.T. 1920 The pigmented epithelium of the embryo chick’s eye studied
in vivo and in vitro. Johns Hopkins Hosp. Bull., vol. 31.

Tart, Jonn 1918 Capillary phenomena observed in blood cells, thigmocytes,
phagocytosis, amoeboid movement, differential adhesiveness of cor-
puscles, emigration of leucocytes. Quart. J. Exper. Physiol., vol. 12.

UnsuenuouTa, E. 1914 Cultivation of the skin epithelium of the adult frog,
Rana pipens. J. Exper. Med., vol. 20.
1915 The form of the epithelial cells in cultures of frog skin, and
its relation to the consistency of the medium. J. Exper. Med., vol. 22.

PLATE 1
EXPLANATION OF FIGURES

1 Culture 708. Subcutaneous tissue, 8-day chick embryo; 2-day culture;
pH 7.6; janus green, iodine. X 146.

2 Same culture. X 480.

3 Culture 667. Endothelium from liver, 7-day chick embryo; 3-day culture;
janus green, iodine. X 146.

4 Same culture. X 480.

5 Culture 666. Endothelium from liver, 5-day chick embryo; 4-day culture;
pH 6.5; janus green, iodine. X 146.

6 Same culture. X 480.

50

ENDOTHELIUM IN TISSUE CULTURES PLATE 1
> WARREN H. LEWIS

Ga.

¥

f

51

PLATE 2
EXPLANATION OF FIGURES

7 Culture 681. Endothelium from liver, 8-day chick embryo; 7-day culture;
janus green, iodine. X 146.

8 Same culture. X 480.

9 Culture 636. Endothelium from liver, 10-day chick embryo; 1-day cul-
ture; janus green, neutral red, iodine. X 480.

10 Culture 678. Endothelium from liver, 8-day chick embryo; 4-day cul-
ture; janus green, iodine. X 480.

11 Culture 717. Endothelium from liver, 8-day chick embryo; 6-day cul-
ture; pH 7.4; janus green, iodine. X 480.

12 Culture683. Endothelium and liver cells from liver, 8-day chick embryo;
6-day culture; pH 6.4; janus green, neutral red, iodine. X 480.

52

“PLATE 2

ENDOTHELIUM IN TISSUE CULTURES

WARREN H. LEWIS

53

PLATE 3
EXPLANATION OF FIGURES

13 Culture 636 (same culture as fig. 9). Endothelial cell from mid part of
outgrowth. Liver, 10-day chick embryo. 1-day culture. Mitochondria,
threads, rods, granules; few neutral red granules. Janus green, neutral red,
jodine. X 1450.

14and15 Culture 717 (same culture as fig. 11). Endothelial cells from liver,
8-day chick embryo; 6-day culture; pH 7.4 Variations in shape of cells and
arrangement of mitochondria. Janusgreen,iodine. X 1450.

54

PLATE 3

ENDOTHELIUM IN TISSUE CULTURES

WARREN H. LEWIS

Sesiciundstiedsatonbeaeacieaaicaeretereeeee

et ae ae

50

PLATE 4
EXPLANATION OF FIGURES

16 Culture 613. Endothelial cell from liver, 7-day chick embryo; 5-day cul-
ture. Mitochondrial vesicles, degeneration vacuoles, large centrosphere,
cytoplasmic striae. Janus green, neutral red, iodine. X 1450.

17 Culture 612. Endothelial cell from liver, 9-day chick embryo; 3-day
culture. Marked striation of cytoplasm. Janus green, neutral red, iodine.
x 1030.

18 Culture 716. Endothelial cell from liver, 8-day chick embryo; 6-day
culture; pH 7.4. Branching mitochondria. Janus green, iodine. X 1450.

19 and 20 Culture 666 (same culture as figs.5 and 6). Endothelial cells from
liver, 5-day chick embryo; 4-day culture. Fig. 19, mitochondrial granules
and vesicles, large centrosphere. Fig. 20, mitochondrial threads, rods,
granules, large centrosphere. Janus green, iodine. X 1450.

56

ENDOTHELIUM IN TISSUE CULTURES PLATE 4

WARREN H, LEWIS

«adler op eee Pb Oe 5"
>

PLATE 5
EXPLANATION OF FIGURES

21 Culture 675. Endothelial cells from liver, 7-day chick embryo; 7-day
culture. Numerous large degeneration vacuoles, mitochondrial granules,
large centrosphere Janus green, iodine. X 480.

22 Culture 694. Endothelial cells from liver, 7-day chick embryo; 7-day
culture. Large vacuoles (dark in figure), pale long mitochondria. Janus
green, neutral red, iodine. X 1450.

23 and 24 Culture 676. Endothelial cells from liver, 6-day chick embryo;
10-day culture; pH 6.6. Fig. 23, fragmentation of nucleus, vacuoles, mitochon-
drial threads, rods, granules. Fig. 24, extrusion of nucleolus. Janus green,
iodine. X 1450.

ENDOTHELIUM IN TISSUE CULTURES PLATE 5
WARREN H. LEWIS

Resumen por el autor, Otto F. Kampmeier

El desarrollo de los linfaticos anteriores y corazones linfaticos
de los embriones de los anuros

El orfgen y desarrollo del seno linfatico maxilar primario,
lifaticos yugulares y corazones linfaticos anteriores de embriones
de sapo es objeto de descripcién en el presente trabajo. El
seno se origina a expensas de pequenos esbozos discontinuos que
aparecen como engrosamientos del endotelio de las yugulares
externas en vias de desarrollo 0 como islotes en el mesenquima
que rodea a las anteriores. Al principio macizos, estos esbozos
adquieren una cavidad y mediante proliferacién se alargan, se
funden, ramifican y forman de este modo una red complicada.
Esta red se transforman en un reservorio espacioso mediante
expansion de los canales interanastomédticos y reduccién de los
cordones mesenquimdticos que los separan. El] linfatico yugu-
lar se desarrolla a expensas de un plexo venolinfatico que de-
riva de las tres primeras venas intersegmentarias (tributarios
dorsales del seno venoso pronéfrico), cuando estas se separan
del sistema venoso. Después el linfatico yugular establece
continuidad con el seno linfatico maxilar primario y con el cora-
zon linfatico anterior.

El] coraz6n linfatico anterior se origina en una porcidn circun-
serita del plexo veno-linfatico, mencionado anteriormente, al
nivel de la tercera vena intersegmentaria original. El esbozo
plexiforme se desarrolla en la cimara cardiaca por distensién y
fusion de sus canales reunidos. ‘Temporalmente se aisla del
plexo veno-linfatico cireunyacente, pero persiste unido con
las venas en la boca de la tercera vena intersegmental, de la
cual deriva la vena vertebral anterior. Las comunicaciones
entre el corazén linfatido y los linfaticos aferentes se reestablecen
ulteriomente. La formacion de las valvulas y la histogénesis de
las paredes del corazon son también objecto de descripeibn.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 28

THE DEVELOPMENT OF THE ANTERIOR LYMPHATICS
AND LYMPH HEARTS IN ANURAN EMBRYOS!

OTTO F. KAMPMEIER
Department of Anatomy, College of Medicine, University of Illinois, Chicago

THIRTY-FIVE FIGURES INCLUDING EIGHT COLORED PLATES”

THE LYMPHATIC GROUND-PLAN OF THE TADPOLE

As is well known, the vessels which collect the lymphatic fluid
and convey it to the lymph hearts and thus to the veins in the
fully developed anuran Amphibia are in the form of extensive
subcutaneous sacs and deep sinuses. This condition, however,
is a relatively late acquisition in development, appearing during
the metamorphosis of the individual. Before this period, the
lymphatic conduit system consists of narrower ducts and capil-
lary networks, similar to those found in the higher vertebrates.
In fact, we can recongnize three periods in the development of
the lymph channels in Anura: firstly, the initial formative period,
secondly, a phase of specific ducts and plexuses, and, thirdly,
the final condition, characterized by broad lymph sacs and sin-
uses—periods which in a general way coincide with the three
into which we arbitrarily divide the embryogeny of frog and toad,
namely, early embryonic, larval or tadpole, and metamorphic
phases. To follow intelligently the nature of events which occur
during the formation of the anuran lymphatic system fromits
inception to its final configuration, as well as to emphasize par-
ticular components and to propose a terminology which will facili-
tate comparison with other vertebrates, it seems expedient to

1The present communication represents a portion of a monograph intended
for publication in 1918. Other papers which will follow complete the subject-
matter of this monograph. The reason why it was broken into a number of
separate parts is explained in a footnote of the first installment which appeared

in The Anatomical Record, vol. 19, July, 1920.
2 The cost of illustrations in part borne by the Anatomical Laboratories.

61

62 OTTO F. KAMPMEIER

delineate the topography of the lymphatics functional in an
intermediate stage.

In Bufo tadpoles,* 12 to 15 mm. long, the chief lymph vessels
are already laid down, so that we may say the second phase of
lymphatie organization, suggested above, begins at this time.

31 spent the spring and summer of 1913 with great profit and pleasure at the
Anatomical Institute of the University of Munich, where, through the kindness
of Professor Riickert, I was able to enjoy all the facilities of those delightful
laboratories. I wish also to mention Dr. H. Marcus, who placed his series of
larval Gymnophiona at my disposal. Further, I express my gratitude to Mr.
Otto Balbach, of the University of Pittsburgh, who prepared the later series of
my numerous sectioned anuran embryos.

Toad embryos constitute by far the bulk of the material used in the investiga-
tion. These specimens are of two species, the American common toad, Bufo
lentiginosus, and the European, Bufo vulgaris (?), and are respectively from
New Jersey and Wisconsin, and from the marshes along the Isar River near
Munich. The writer neglected to determine with absolute certainty the specific
name of the latter form before leaving Munich. There are but two species which
can be considered, Bufo vulgaris and B. viridis, but after comparing them as to
distribution, breeding habits, etc., as described in the standard works of Zoology,
he is confident that it is Bufo vulgaris. The descriptions and figures are based
mainly on the embryos of this European form which were gathered later in the
course of the investigation when the writer had attained greater success in the
preparation of tissues so profusely filled with yolk as are amphibian embryos.
The ova of these Anura were collected shortly after laying and developed in the
laboratory aquaria. To procure a closely graded ontogenetic series, active
individuals were fixed and preserved at intervals of three to four hours.

The ordinary methods of technique were employed in the preparation of the
serial sections. Before the embryos exhibited movement, they were fixed directly
in Zenker’s fluid, but later embryos were first anesthetized in a weak chloretone
solution to prevent the distortion or tearing of the delicate tissues which might
result from the writhing or twitching of the body when placed in the irritating
fixative. The difficulties at first encountered in making satisfactory serial
sections, apparently due to the brittleness of the yolk-laden tissues when xylol
was used as the clearing reagent, were overcome by using cedar oil instead and
by diminishing the time of paraffin infiltration to a minimum. Both the graphic
method and the modified Born’s wax-plate process were enlisted in the execution
of the reconstructions. In every case, the outline drawings of the sections were
made with the Edinger projection apparatus. The writer also attempted to
inject the vascular channels of a number of embryos, but on the whole he met with
little success. The continuous layer of brown pigment in the skin of the toad
larvae hides the underlying structures, and the cannula needle can therefore not
be directed with the same degree of certainty as when transparent fish embryos
or the much larger pig or chick embryos are injected under the binocular micro-
scope.

eire.or.si.
mox.prim.

cire.or.si.
méx.prim.

V.jug.exf.
Sin.

Fig. 1 Transverse section of a 13-mm. embryo of Bufo vulgaris at the level
of the mouth. X 50. circor. si. max. prim., circumoral division of the primary
maxillary sinus; cav. or., cavum oris; lab. or., labial structures of the larval suck-
ing mouth.

Fig. 2 Same, at the level of the eyes. X 50. mand. si. max. prim., mandi-
bular division of sinus lymphaticus maxillaris primigenius; v. jug. ext. dex. and
sin., vena jugularis externa dextra and sinistra; a. car. ext. dex. et sin., arteria
carotis externa dextra and sinistra; cav. or., cavum oris.

63

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 1

64 OTTO F. KAMPMEIER

The most marked feature of the disposition of the lymphatics
in the head is the relatively enormous expanse of a lymph sinus
situated in the ventral and lateral cephalic territory. Its limits
in a 26-mm. frog larva are shown in Hoyer’s sketches, illustrated
in Wiederscheim’s ‘Vergleichende Anatomie der Wirbeltiere’
(7th et al. editions), and in the wax reconstruction of the vascular
channels in the head of a toad embryo in figure 28. This lymph
reservoir, which the writer designates the primary maxillary
sinus (sinus lymphaticus primigenius maxillaris)? because eventu-
ally it is resolved into the secondary lymph sinuses in the region
of the jaws, is developed very early and, in general form, extent
and proportions, is virtually complete in 9- or 10-mm. toad
embryos, hence, at a period when most of the other lymphatics
are still in the formative state. Figure 28 shows that this sinus
does not possess a simple contour, but is composed of several
interconnecting chambers of diverse shape and size. For con-
venience and clearness, we may refer to the several subdivisions
by different names. The broad, roughiy rectangular division
(figs. 2, and 28, mand. si. max. prim.) on the ventral side of the
head may be regarded as the mandibular one; it is the largest,
the first to develop, and the other portions of the sinus arise
from it by outgrowth and extension. In continuity with it
anteriorly is the cireumoral division (figs. 1 and 28, circ. or si. max.
prim.), which encireles the mouth opening. The third division,
a pair of temporal chambers (figs. 3 and 28, temp. si. max. prim.),
appears in the wax model as two lateral wing-like expansions of
the mandibular sac; these extend as far as the pronephroi, where
each contracts into a narrow duct which leads to the anterior
lymph heart of the respective side. ‘The fourth division of the
primary maxillary sinus may be termed the pericardial (figs. 3 and
28, pericard. si. max. prim.); it constitutes a second path of
communication between the mandibular and temporal sacs, but
at a deeper level. It is paired and branches, as a more slender
and somewhat plexiform channel, from the mandibular sac near

4 Hoyer calls this the ‘Kehlsack’ and Jourdain ‘sac gulaire,’ and in my paper on
the origin of the lymphatics in Bufo (’15) it is spoken of as the ventral cephalic
sinus, but these terms are too general.

DEVELOPMENT OF LYMPHATICS IN ANURA 65

its posterior margin, but in its course it curves dorsally, that is,
centrally or inwardly, and, closely associated in position with
the external jugular vein, passes back along the heart towards

temp.si.mox.
prim.
Ly
hy.
rg
eq
Wf
ef)
or|\
Hy
Ff
RY
* iy
a.
‘al
aN
=
ae ;
\ oe NS f Be t ee rs
Be B V4 }S si. FX. prim eae
Weed i oe pericord. gf | ae
MT Aa i Si.ven. 31.MOX. Pa y" ie
*% 4 S ae oa i ke 7 SE fy sy ay
\ ~ cs ;
AUC a mG I AY ‘
Maar 7 lh : é Z
eS aa 2 int.
ia ; da WW ig
ee 4
SOG ae ae KE
r ae q A ae
Le oe a SE
NA Low pete ~N
GA Oe ens SS
oe é4 ripe SS
: 2s 23 PS —
wa oe it renee
8 SI 8 oP?
SS ae

Fig. 3 Same, at the level of the auditory capsule. X 50. temp. and peri-
card. st. max. prim., temporal and pericardial divisions of the primary maxillary
sinus; v. jug. int. and ext. dex. and sin., venae jugulares internae and externae
dextrae and sinistrae; sz. ven., sinus venosus; cav. phar., cavum pharyngeus; cav.
bran., cavum branchialis; int., intestinum; coel., coelom.

the sinus venosus and thence makes a broad sweep outward
along the duct of Cuvier to join the hinder end of the temporal
sac.

66 OTTO F. KAMPMEIER

lym. jug.
0 nephst.
proneph.

cor. lym. ant.
proneph.

Fig. 4 Same, at the level of the anterior limb bud (brach.).. X 50. lym. jug.,
lymphatica jugularis; proneph., pronephric tubules and sinusoids; JI nephst.,
2nd nephrostome; glom., pronephric glomerulus; int., intestinum; oesoph., oeso-
phagus; ves. fel., vesica fellea; hep., hepar; coel., coelom.

Fig. 5 Same, at the level of the 3rd spinal ganglia. X 50. cor. lym. ant.,
cor lymphaticum anterius; vent., venter. Other references as in the preceding
figure. Lying medial to the pronephric tubules and sinusoids (proneph.) are, in
the order named, the primary excretory duct, the subeardinal vein and the aorta.

DEVELOPMENT OF LYMPHATICS IN ANURA 67

The primary maxillary sinus receives the lymphatic drainage
of the head, as indicated in Hoyer’s sketches. The lymph then
flows posteriorly towards the anterior lymph heart of the same
side through the channel (fig. 28, lym. jug.), which has been
mentioned as a caudal prolongation of the temporal portion of
the sinus, though genetically it has an independent origin. Hoyer
has called this vessel the ‘cephalic duct’ or ‘ Kopfgefiiss,’ but the
term jugular lymphatic (lymphatica jugularis)> seems more
appropriate on account of its probable homology with a similar
vessel in all other vertebrates. It lies immediately dorsal to the
pronephros (fig. 4) and only a short distance below the skin.
Near the lymph heart a tributary is given off which extends to the
anlage of the forelimb, at this time a knob-like condensation of
mesenchyme beneath the operculum, and, as it is the parent
of the future lymph vessels of the arm, this tributary may be
called the brachial lymphatic (lymphatica brachialis).

The relative size, shape, position, and connections of the pair
of anterior lymph hearts during the second embryonic phase are
exhibited in the wax reconstruction (fig. 28) and in the section
of a 13-mm. embryo (fig. 5). Each heart is globular in form,
placed superiorly at the posterior limit of the pronephros and is
in continuity with both vein and lymphatic duct. It is located
in the triangular area, bounded by skin, myotome, and the roof
of the coelom at the level of the third spinal ganglion. On its
ventral side it opens at the junction of the pronephric sinus and
a short dorsal venous extension, the rudiment of the anterior
vertebral vein of the adult. In the opposite wall of the heart the
afferent lymphatic vessel has its entrance.

Not only the lymphatic drainage of the head is poured into the
anterior lymph hearts, but also the greater quantity of the lymph
from the trunk is conveyed to them by two pairs of important
ducts, the subvertebral lymphatics (lymphaticae subvertebrales),
lying deep, and the lateral lymphatics (lymphaticae laterales)
of the trunk situated superficially, one on each side. The latter

> The usage of the word ‘lymphatic’ and its Latin form, ‘lymphatica,’ has been

made clear in the author’s paper in The Anatomical Record, vol. 16, no. 6, August,
1919.

lym. subvert.

a
iS
ey

\ym subvert.

Jat.v.card.post.

lat v.card. post:

a

es
Bion

AN cor lym. post.

OF.

DEVELOPMENT OF LYMPHATICS IN ANURA 69

are a direct continuation backwards of the jugular lymphatics,
as illustrated in figure 28, and together they possess a common
opening into the lymph hearts. In its course caudalward, each
lateral lymph duct runs between the myotomes and the epidermis
(fig. 6, lym. lat.), in the lateral-line region. About halfway
towards the posterior lymph heart, it sends off a branch which
passes over the upper edge of the myotome to fuse with its fellow
of the opposite side and assuming a median position (fig. 7,
lym. dors.), proceeds distally into the tail as the dorsal lymphatic
(lymphatica dorsalis).

The paired subvertebral lymphatic, corresponding to the tho-
racic ducts of the higher vertebrates, has a position along the
aorta and dorsal to the postcardinal veins. Anteriorly, it curves
outward under the lower margins of the myotomes to join the
anterior end of the lateral lymph ducts in the immediate neigh-
borhood of the anterior lymph hearts. It retains the axial loca-
tion (fig. 6, lym. subvert.) throughout almost its entire extent and
later becomes connected with its companion by occasional anasto-
moses. In the vicinity of the posterior lymph heart, the duct
again bends outward to reunite with the lateral lymph vessel of
the same side (fig. 7). The common duct so formed later com-
bines with the opposite one in the ventral midline and is pro-
longed caudally into the tail as the ventral caudal lymphatic
(lymphatica ventralis) in the base of the ventral tail fin. In
this region, too, the iliac lymphatic (lymphatica iliaca) is given
off to the hind limb bud.

Fig. 6 Transverse section of a 15-mm. embryo of Bufo vulgaris through the
trunk at the level of the 9th spinal gangha. lym. lat., lymphatica lateralis; this
duct is plexiform in character; lym. subvert., lymphatica subvertebralis (tho-
racic duct); 8 v. seg., 8th intersegmental vein; lat. v. card. post., lateral division
of the posteardinal vein; the medial divisions (subeardinals) have fused to form
the posteava which lies ventral to the aorta; mesoneph., mesonephric tubules and
sinusoids; rect., rectum; int., intestinum; coel., coelom.

Fig. 7 Same, at the level of the 11th spinal ganglia. X 50. cor lym. post.,
cor lymphaticum posterius; lym. dors., lymphatic dorsalis; 11 v. seg., 11th inter-
segmental vein. Other references as in the preceding figures. Medial to the
subvertebral lymphatics are the aorta, primary excretory ducts and the post-
cardinal veins.

70 OTTO F. KAMPMEIER

The paired posterior lymph heart is similar in shape to the
anterior, though somewhat smaller in size at this period (15-mm.
embryo), and lies lateral to the myotomes in the intersegment
of the 11th and 12th (fig. 7, cor. lym. post.). It joins the 11th
intersegmental vein (1/1 v. seg.) which becomes, as shown pre-
viously,® the proximal portion of the posterior vertebral vein.
The heart receives the lymph stream from the hinder regions
of the trunk and the tail through the lateral lymph duct.

All of the main lymphatic conduits described possess sub-
sidiaries and capillary plexuses, the ramifications of which in
frog larvae are admirably shown in injected specimens, as illus-
trated by Hoyer.

In the present paper, the origin and development of the pri-
mary lymph sinus, the jugular lymphatics, and the anterior lymph
hearts will be considered. The formation of the lymphatics of
the trunk and tail, including the posterior lymph hearts, will be
taken up in a succeeding article.

THE DEVELOPMENT OF THE PRIMARY MAXILLARY LYMPH SINUS’

In 5-mm. embryos (Bufo vulgaris) a crude vascular plexus
exists ventral to the oropharyngeal cavity and has its greatest
concentration in the vicinity of the thyroid diverticulum. From
this plexus the external jugulars’ and external carotids and their
tributaries subsequently differentiate. But at this time veins
and arteries are still broadly confluent; all channels are alike in
histological appearance, and merely the definite and constant
position of certain ones enables us to pick out the future arterial
and venous components. Farther back towards the heart, how-
ever, a division has already occurred between them, and the
external jugulars and carotids are independent, the former curving
laterally around the ventricle to join the common cardinal veins

6 Anatomical Record, vol. 9, July, 1920.

7 A short description of the genesis of the primary lymph sinus in the head of
Bufo embryos was published by the writer in The American Journal of Anatomy,
vol. 17, 1915.

8 In using the term ‘external jugular vein’ the author is following Gruby and
Ecker; Goette and many other authors refer to this vein as the ‘inferior jugular.’

DEVELOPMENT OF LYMPHATICS IN ANURA el

and the latter being in continuity with the aortic arches. In
the reconstruction reproduced in figure 29 their topographical
relations are clearly indicated, though this deals with a later
stage, a 6-mm. embryo, in which the demarcation between
jugular (v. jug. ext.) and carotid (a. car. ext.) is complete except
anteriorly, where they are still in broad plexiform connection.

The inception of the primary maxillary sinus takes place in
5-mm. embryos during the period of the indifferent jugulocarotid
plexus, just described. Its initial anlagen arise along those
channels which are to become the external jugular veins, and at
first many of them are in the form of short knot-like cellular
thickenings adhering to their lining. Such alymphatic anlage is
shown in the photomicrograph, figure 8, as a compact protu-
berance (lym.) of the intima of the blood vessl (v. jug. ext. dex.).
A transverse section of another sinus anlage of the same speci-
men, but from the opposite side, is pictured in figure 9, 6. In
longitudinal extent, it passes through seven sections (each 6 u
thick). It is a solid cell cord or column attached to the wall
of the vein (v. jug. ext. sin.) by its anterior end, while throughout
the remainder of its course it lies free in the mesenchyme ventral
and parallel to this vessel.

Besides the adherent lymphatic anlagen, there are at this stage
other anlagen, which, though they be similar to them in size,
shape, and location, are not in immediate contact with the lining
of the blood channel, and the question naturally arises: Were
such anlagen formerly connected with the haemal endothelium,
or did they arise independently? Observations on the succeeding
genetic stage, as well as the investigation of the developing
lymphatics of the trunk region, furnish evidence that points to
the independent origin of such anlagen and besides reduces the
significance which we would attach to the adhesion of some of
the earliest lymphatic anlagen to the primitive blood channels.
The theoretical aspects of this problem will be discussed after
the steps in the development of the primary maxillary lymph
sinus have been described.

The lymphatic anlagen, like the endothelium of all blood
channels, especially in the head region during early development,

fl OTTO F. KAMPMEIER

are stuffed with large yolk globules from tip to tip—a fact that
clearly distinguishes them from the surrounding mesenchymal
cells which have for the most part lost their yolk content. The
nuclei of incipient endothelium, regardless of whether haemal or
lymphatic, show no difference, except possibly in chromatic
density when compared with those of mesenchyme; indeed, the
endothelium presents a very unspecialized appearance. The
fact of the longer retention of yolk spherules by the cells of vas-
cular anlagen and channels was reported by the author (15) and
emphasized as a diagnostic trait of considerable value in dis-
criminating between these tissues during the earlier embryonic
period. Their distinctions were accurately expressed in the
colored figures of that paper, to which the reader is referred.

The next older stage, a 6-mm. embryo, is characterized by the
numerical increase of sinus anlagen along the external jugular
veins, by their growth in length and budding of branches, their
detachment from the venous intima at the original point of con-
tact, and by their acquisition of lumina. The reconstruction in
figure 29 displays the number, size, form, affinities, and distri-
bution of these lymphatic anlagen. It furnishes a convincing
picture to show that the sinus does not originate by centrifugal
sprouting from any specific foci, but that it has a multiple origin
in proximate relation with vessels of the primitive vascular net-
work, and that the initial anlagen are discontinuous. Further,
it shows the bilateral origin of the sinus and also that its principal
or mandibular division is the first component to be formed, the
other divisions, such as the circumoral, temporal and pericardial
appearing somewhat later.

The time at which the individual lymphatic anlagen that are
adherent to the venous wall retract from it varies greatly, neither
the time of their beginning nor their length entirely conditioning
it. In the reconstruction (fig. 29) some of them still cling to the
blood channel, while others, even smaller ones, lie independently
in the surrounding tissue. Nor is the size of the anlage a cri-
terion of the possession of a lumen; one may acquire such very
early, even in its incipient stage, while another may remain solid
for a longer period of time. When a lumen does appear, it is at

DEVELOPMENT OF LYMPHATICS IN ANURA 73

7 |v. jug.ext. sin.

atin mee AOE be Maes B | lym. ae ie
Fig. 8 Photomicrograph of a transverse section through the right ventral
cephalic region in a 5-mm. embryo of Bufo vulgaris (Kampmeier Embryological
Collection, series B 25, slide 1, section 71). X 690. (Zeiss Apochromat. Obj. 4
and Compensat. Project. Oc. 4,). ep. epidermis; v. jug. ext. dex., vena jugularis
externa dextra; lym., and initial lymphatic anlage of the primary maxillary
sinus; the structure lying within the upper right-hand portion of the lumen of
the vein is a yolk-filled blood cell. In this and the following photographs, the
yolk globules can be easily distinguished from the dense cell nuclei by their
smaller oval shape and their uniform gray color.

Fig.9 Photomicrograph of transverse sections through the left ventral
Cephalic region in a 6-mm. embryo of Bufo vulgaris (K. E. C., series B 54, slide 1,
Sections 75 (A) and 80 (B). X 690. v. jug. ext. sin., vena jugularis externa sin-
istra; lym., initial anlagen of the primary maxillary sinus.

74 OTTO F. KAMPMEIER

V.JUS.€xT. dex.

Fig. 10 (A) Photomicrograph of a transverse section through the right
ventral cephalic region of a 6-mm. embryo of Bufo vulgaris (IX. E. C., series B 53,
slide 1, section 90). X 690. (B) Section through the left ventral cephalic
region of a 7-mm. embryo (series B 52, slide 1, section 84). XX 690. v. jug. ext.
dex. and sin., vena jugularis externa dextra and sinistra; lym., anlagen of the
primary maxillary sinus.

aE

DEVELOPMENT OF LYMPHATICS IN ANURA 0

first a cleft or vacuole in the cytoplasm’ between the large pro-
minent yolk globules (fig. 10, A), or if the anlage be larger, it
may consist of a number of crevices which soon coalesce to pro-
duce a more conspicuous cavity, (figs. 9. A, and 10). The lumen
naturally expands with the growth of the anlage, but during
several successive stages the confines of these lymphatics, like
those of the haemal vessels, remain irregular and of varying
thickness and appear gnarled, particularly in section (fig. 10),
owing to the groups of large ovoid yolk globules which they con-
tain and which do not entirely vanish until a relatively late
period of sinus formation.

From now on the development of the smus makes rapid prog-
ress. The discrete lymphatic anlagen of the same side establish
continuity with one another by end-to-end fusion and begin to
send out endothelial extensions in a ventromedial direction.
These sprouts actively proliferate, branch and rebranch, and
freely anastomose with one another in such a way as to produce
a plexus, the meshes of which all lie in the same plane. As the
identical condition prevails on the opposite side of the head, the
two plexuses approach each other, meet and combine in the
midline and so create a broad intricate network (fig. 11, from
lym. to lym.) extending in a curved plane from the vicinity of
one external jugular to that of the other through the loose mes-
enchyme between the thyroid and the epidermis on the ventral
surface of the head. This network is the anlage of the principal
or mandibular division of the primary maxillary sinus and is
shown in the reconstruction in figure 30 (sz. mand.). In the
drawing, the vascular channels are pictured in a flat plane, though
in reality the most distal structures bend dorsolaterally. It may

* The formation of the lumen, as indicated, raises the question, whether it is
of intracellular or intercellular origin. The answer rests partly on our definitions
of ‘cell’ and ‘syneytium.’ Are the spaces of mesenchyme to be considered as
Sntercellular’ or ‘intracellular’? The originally solid lymphatic anlagen, de-
scribed above, are probabry, like other mesenchymal tissue, syncytial in nature,
and accordingly I would look upon the vacuole-like beginnings of their lumen as
being intracellular in situation. Subsequently, with the expansion of the lym-
phatic anlage into a definite vessel and the appearance of distinct cell boundaries
in its endothelium, the lumen acquires its intercellular character.

76 OTTO F. KAMPMEIER

v.jug.ext.dex. 1 &.car. ext. dex.) &. cat ext. sin.

lym.

: acat: ext. dex.et sin. :

«> at oS ;
yay ~~ my : s 1 \ym. i, 12, ‘ A nt Fe *- “s ay | lym. Pet %,

Fig. 11 Photomicrograph of a transverse section through the ventral cephalic
region in a 7-mm. embryo of Bufo vulgaris (K. E. C., series B 27, slide 1, section
100). X 290 (Leitz 4-mm. Obj. and Zeiss Compensat. Project. Oc. 4). a. car.
ext. dex. and sin., arteria carotis externa dextra and sinistra; v. jug. ext. dex.,

DEVELOPMENT OF LYMPHATICS IN ANURA 77

be noted that the outer limit of the principal or mandibular
plexus is sharply defined by a pair of broader, longitudinal vessels
which genetically represent the oldest portion. In this stage (a
7-mm. embryo) also the anlagen of the other divisions have made
their appearance as outgrowths from the principal one. Anteri-
orly, the circumoral division (s?. circor.) 1s an extension, on
either side, growing forward along a ring-like vessel which is a
branch of the external jugulars and encircles the mouth opening.
Eventually the two halves of this division, by further elongation,
meet and unite in front of it. Posteriorly, the pericardial di-
vision (si. pericard.) of the sinus consists of a pair of caudally
directed extensions, closely accompanying the external jugular
veins towards the sinus venosus, where in subsequent stages they
become prolonged outward to join the terminal portion of the
temporal division of the same side. In the reconstruction under
consideration each temporal division (s?. temp.) is shown as a
derivative of a slender lymph vessel which extends laterally
around the oropharyngeal cavity in association with a tributary
of the external jugular vein. At this time the temporal division
has already made considerable advance in plexus formation, but
further tips continue to proliferate and to anastomose. One of
these offshoots, passing back in the broad expanse of loose tissue
lateral to the aortic arches, is highly distended locally (fig. 30),
a condition manifestly produced by the pressure of the lymph
collected within its lumen. A similar saccular enlargement of
the temporal plexus exists also on the opposite side. In several
sections on the right side, the writer was unable to follow with
certainty its connection with the remaining part of the plexus,
and on the reconstruction this point has been indicated by an

vena juglaris externa dextra; the heavy masses in the center of the figure are mus-
cle anlagen; in a curved line from lym. to lym., sections of the interanastomosing
channels of the primary maxillary lymph sinus during its plexiform stage (cf.
fig. 30).

Fig. 12 Photomicrograph of a transverse section through the ventral ceph-
alice region in an 8-mm. embryo of Bufo vulgaris (KX. E. C., series B 49, slide 1,
section 88).- X 290. lym., the channels of the plexiform primary maxillary
sinus are beginning to coalesce with one another by their expansion. Other
references as in figure 11.

7 OTTO F. KAMPMEIER

interrogation mark. It is probable that during the fixation of
the embryo, the very slender connecting channel had collapsed
or contracted into such a delicate strand that it became im-
possible to distinguish it from the surrounding mesenchymal
reticulum.

The reconstruction (fig. 30) shows that the primary outgrowths
of the circumoral, temporal, and pericardial divisions from the
principal plexus keep close to haemal vessels, potential veins.
In fact, frequently, and particularly in the case of the pericardial
division, the lymphatic extension adheres to the wall of the
blood vessel. The writer has been unable to decide whether or
not, in the elongation of such lymph channels, the endothelium
of the blood vessel contributes cells to the growing tip. It is
conceivable that the latter might simply advance along a path
of least resistance or in accordance with certain stresses or cur-
rents that may closely parallel the blood vessel. As yet we are
entirely ignorant of the presence or absence of any such pro-
nounced currents in the tissue interstices before the advent of the
haemal and lymphatic capillary systems, and the suggestion that
the paths invariably taken by these primary lymphatic exten-
sions may be predestined by the existence of definite antecedent
streams, acting as a stimulus or directive force to the prolifera-
ting endothelium, is pure conjecture. A cross-section of the
pericardial division illustrating the adhesion of the lymph vessel
to the haemal one is shown in figure 14 (lym. and v. jug. ext. sin.).

During the formation of the plexus phase of the primary maxil-
lary sinus, the sprouts and the most recently established anasto-
moses are usually solid, the acquisition of lumina, however,
occurring very soon. During this period, too, the number of
yolk spherules in the lining cells are still very abundant.

The next phase in the development of the lymph sinus is the
transformation of the plexus into a spacious and uninterrupted
chamber. This process is a rapid one, being practically finished
in the embryonic period between 8- and 10-mm. stages (B. vul-
garis). The genetic changes consist in the progressive expan-:
sion of all the anastomosing channels, so that the gaps in the net-
work are reduced and the mesenchyme filling them is compressed

DEVELOPMENT OF LYMPHATICS IN ANURA 79

into trabeculae, which become more and more attenuated, and
finally break and disappear as the sinus becomes more greatly
distended in its vertical, that is, dorsoventral, diameter. ‘These
successive steps are clearly exhibited in the inserted photomi-
crographs, figures 11, 12, and 13, the last two illustrating how
the mesenchymal strands are drawn out and tear and how their
remnants persist for a time as longer or shorter spurs which pro-
ject into the sinus cavity.

During the further growth and enlargement of the sinus, I
was unable to find the addition of separate mesenchymal spaces
by concrescence, such as I described in the development of the
thoracic duct in the pig (’12) or those of McClure (15) in the
formation of the subocular lymph sac in the trout, or those of
Huntington (11) on the growth of the periaortic lymphatics in
Chelydra.

I ean but believe that the coalescence of the originally dis-
continuous lymphatie anlagen, the formation of the intricate
lymphatic plexus and its conversion into the relatively enormous
sinus is largely, perhaps wholly, due to the accumulation within
their lumen of lymph, which, as it increases in quantity, increases
the internal pressure on its walls and achieves the extension and
distention of the developing sinus, for during this important genetic
period the sinus possesses no outlet; it is not confluent with the veins.
The saccular and expanded posterior prolongations of the tem-
poral plexus shown in the reconstruction (fig. 30) certainly point
to such an interpretation. A similar view was expressed by
McClure (’15) in his preliminary paper on the development of
the anterior lymphatics in teleost embryos.

Coincident with the expansion of the lymph sinus, its lining
cells assume all of the attributes of typical endothelia. The cells
become much flattened, and their nuclei, which in earlier stages
resembled those of mesenchyme in their spherical shape and their
coarse chromatic texture, become more and more compact and
dense like the intimal nuclei of older vascular channels. The
yolk corpuscles in the cytoplasm of the endothelium also gradu-
ally disappear, although in 9- and 10-mm. embryos a few are
still to be found.

THE AMBRICAN JOURNAL OF ANATOMY, VOL. 30, No. 1

80 OTTO F. KAMPMEIER

In embryos, approximately 10 or 11 mm. long, the primary
maxillary sinus acquires an outlet. The posterior extremity of
the temporal division by further backward prolongation (figs. 34
and 35) becomes confluent with the jugular lymphatic, which in
turn gains access to the anterior lymph heart and conveys thither
the lymph collected by the sinus.

During the later larval and metamorphic periods, the primary
maxillary sinus, as well as the other lymph channels laid down
in the embryo, are converted into the superficial and deep lymph
saes found in the adult.!°. Such changes will be reserved for a
later paper.

From the foregoing account of the appearance and relations of
the early adherent anlagen of the primary maxillary lymph sinus,
reinforced by the evidence of the photomicrographs illustrating
it, the conclusion is forced upon one that they are probably deriva-
tives by proliferation from the walls of the external jugular
components of the early unspecialized jugulocarotid vascular
plexus. Formerly (15) I believed that these observations
afforded fairly decisive evidence in favor of the origin of lym-
phatics from venous epithelium, and I suggested tentatively that
certain discontinuous mesenchymal spaces of Amniotes, which
had been described previously as incipient lymphatics, might
have been derived early from neighboring blood channels in a
manner hardly perceptible on account of the absence of any
special differential characteristic in either the vascular intima or
the mesenchyme. But after investigating more thoroughly other
lymphatic channels in anuran embryos, as well as considering the
evidence contained in the mass of literature which has accumu-
lated in recent years on the problem of vasculogenesis, that opin-

0 In The Anatomical Record, vol. 16, 1919, the writer stated that topograph-
ical relations and genetic data show the primary maxillary lymph sinus of anuran
tadpoles to correspond to the subocular lymph sinus of fishes. Since then I have
carried on a comparative study of the lymphatic system in the different classes
of vertebrates, and the available data force me to modify that statement. Pos-
sibly only the dorsal lateral extensions of the principal portion of the primary
maxillary sinus are concerned in the homology. Further observations bearing
on this question will be considered in the comparative anatomy of the lymphatic
system which is in process of preparation.

Fig. 13 Photomicrograph of a transverse setion through the ventral cephalic
region in a 9-mm. embryo of Bufo vulgaris (K. E. C., series B 2, slide 1, section
115). X 290. si..max. prim., primary maxillary lymph sinus; the spurs of tissue
projecting into its lumen are the vestiges of the former bands of mesenchyme
between the lymph channels during the plexus stage of the sinus. Other refer-
ences as in previous figures.

Fig. 14 Photomicrograph of a transverse section through the left ventral
region of the body at the level of the heart in a 7-mm. embryo of Bufo vulgaris
(K. E. C., series B 27, slide 1, section 178). X 690. cor, wall of the heart; cav.
pericard., cavum pericardium; ep., epidermis; v. jug. ext. sin., vena jugularis
externa sinistra; lym., anlage of the pericardial division of the primary maxillary
sinus.

81

82 OTTO F. KAMPMEIER

ion loses weight—a source of gratification to the writer in so far
as he is not compelled to regard his earliest work (12) as funda-
mentally wrong in its deductions. The recent investigations
have brushed away many difficulties, and the interpretation of
the observations rests on firmer ground; views which were thought
conflicting only a few years ago can now be reconciled. The
origin of the earliest vascular anlagen in the embryo is the basic
problem of vasculogenesis, not that of vessels, regardless of their
vascular function, which develop later. If it be true that the
earliest anlagen arise from the mesenchyme, as most of the
modern research on vasculogenesis would indicate, then the two
views of lymphatic development, the venous origin and the
mesenchymal origin of lymph vessels, are not in diametrical
opposition as was formerly vehemently asserted. Indeed, there
may be a number of variations in the genesis of such channels,
but the differences can now be judged superficial, presupposing,
of course, that the haemal and lymphatic systems are not pri-
mordially and phyletically distinct, as some investigators tacitly
hold. Lymphatic anlagen may proliferate from components of
the early indifferent embryonic vascular plexus, or certain channels
may separate from it (just as arteries and veins are differentiated
from it) and assume a lymphatic function; and, again, they may
be formed directly from mesenchyme independently of vascular
channels already existing. What determines the several varia-
tions of lymphatic development is still obscure, although the time
and the site at which they first appear, as conditioned by physio-
logical needs, may be the causative factors. The first method
is illustrated by the origin of the primary maxillary sinus which, with
the exception of the anterior lymph hearts, is the first lymphatic to
appear in Bufo embryos, and perhaps most of the anlagen of
which originate, as has just been described, in connection with
the endothelium of potential haemal vessels before that endo-

11 Anyone wishing to follow the controversy regarding the origin of lymphatics
is referred to the numerous papers which have appeared during the last decade
in America on the problem of lymphatic development. The larger papers of
Sabin, Huntington, and McClure on the development of the mammalian lym-
phatic system contain a comprehensive list of the literature.

DEVELOPMENT OF LYMPHATICS IN ANURA 83

thelium has become specialized, that is, has acquired the attri-
butes of the typical flattened lining cells. The second method
is seen in the formation of the jugular lymph sac of mammals,
and in that of the anterior lymph hearts and the jugular lymph
ducts of the toad. The development of the latter structures
will be described in following sections of this paper, but it may
be stated here that they arise from vessels which, at first, are
freely confluent with the embryonic blood vessels and function
as such, but later separate and become an integral part of the
lymphatic channel system. The third method, the formation of
a lymph vessel by the fusion of mesenchymal spaces, somewhat
like the origin of the earliest vascular channels, is illustrated by
the development of a considerable portion, at least, of the thoracic
duct and other large lymph vessels in mammals, birds, reptiles,
and fishes, as portrayed in numerous papers that have appeared
within the last decade. It was therefore not surprising to dis-
cover this method active also, perhaps solely, in the formation of
the large lymph ducts in Anura which arise later than the anterior
lymph hearts and primary maxillary sinus, at a time when the
blood circulatory system of the embryo had become better
organized and its components more specialized.

THE DEVELOPMENT OF THE JUGULAR LYMPHATIC

Hoyer describes the development of the jugular lymphatic
(cephalic duct) in frog embryos as a centrifugal outgrowth of the
anterior lymph heart, but the writer’s observations show that in
toad embryos, at least, its origin is not so simple.

The jugular lymphaties (fig. 28, lym. jug.), one on each side,
develop at the same time as the anterior lymph hearts and in
the same general region so that they might be discussed together,
though for systematic reasons they will be treated separately.
Figures 31 to 35, inclusive, which illustrate reconstructions of the
important structures in the territory of the left pronephros in
several consecutive stages, furnish a clear idea of the salient and
progressive events that occur. Besides the vascular channels
which are directly and indirectly concerned in the formation of
the lymphatics, other organs, such as the pronephros, spinal

S4 OTTO F. KAMPMEIER

ganglia® and a segment of the neural tube, were introduced in
the reconstructions for the purpose of orientation.

I (20) deseribed the series of intersegmental vessels that
appear in the development of the venous system as dorsal tribu-
taries of the pre- and postcardinal trunks and found them situated
at the intersegments of successive myotomes. ‘The first two
reconstructions (figs. 31 to 32), besides illustrating the beginning
of the anterior lymph heart as a circumscribed plexus of the prox-
imal portion of the third intersegmental, shows the development
of a more open-meshed network of vessels formed by anastomoses
between the first, second, and third intersegmentals. The
jugular lymph duct is derived from the latter plexus. Passing
from the 6- to the 7-mm. stage, the genetic changes consist in
the separation of this intersegmental plexus (which in view of its
former and its future functions may temporarily be called a
venolymphatic) from the precardinal vein and the pronephric
venous sinus. The channels of connection contract in caliber,
like any other small redundant vessel, and finally are cut off
entirely. This is indicated in the reconstruction in figure 33,
where the points marked by a star still show minute and slender
connections, the last traces of the originally freely confluent
condition of the intersegmental veins and their parent trunk.
Farther forward in the figure are two other vestiges in the form
of venous spurs extending towards the lymphatic duct. As the
veno-lymphatie plexus (potentially lymphatic) is severed from
the veins, its channels distend, evidently due to the accumulation
of the lymph within their lumen.

While the foregoing is taking place, a notable event, the tran-
sient isolation of the lymph heart anlage from the surrounding
lymphatic plexus, occurs, a process which will be considered more
fully in the following section. Such a phase is illustrated in the
reconstruction in figure 34. The secondary junction between
heart and afferent lymphatic is brought about a little later. The

2 The first pair of spinal ganglia are evanescent structures in the anourous
Amphibia, being present in toad embryos (B. vulgaris) only during the 6-mm.
stage and vanishing completely very soon after. The second pair become the
first of the adult.

~

DEVELOPMENT OF LYMPHATICS IN ANURA 85

reconstruction exhibits a number of other features. The jugular
lymphatie (lym. jug.) by growth cephalad and the temporal divi-
sion of the primary maxillary lymph sinus (temp. sv. max. prim.)
by growth caudad (figs. 30, 34, and 35) have met and become
continuous. Further, as shown in figure 34, the jugular and the
lateral-line (lym. lat.) lymphatics, united from the beginning,
develop prominent ventral branches lateral to the pronephros.
The other tributaries, extending dorsally and showing a meta-
meric tendency, unquestionably represent the distal portions of
the intersegmental vessels from which the jugular lymphatic was
derived. Finally, the minute connection (starred) between this
plexiform duct and the pronephric sinusoids may be noted, which
has managed to persist until this time. In a later stage (10-mm.
embryo) the jugular, in common with the lateral-line lymphatic,
has reunited with the lymph heart, and farther forward the junc-
tion with the temporal division of the primary maxillary sinus
has expanded (fig. 35).

A few words respecting the venous circulation of the region
under consideration will explain certain difficulties. Since the
anterior intersegmental veins function as haemal conduits before
their transformation into the plexus of the jugular lymphatic, as
soon as their complete separation from the cardinal venous trunk
is accomplished, the region of the myotomes which they drained
would be left without a blood vascular return, but for the develop-
ment of secondary channels from the cardinal veins. Accord-
ingly, such tributaries are laid down at this time, but they are
situated chiefly on the inner suface of the myotomes and accom-
pany the spinal nerves and ganglia; here, besides receiving
branches from the myotomes, they communicate broadly with
similar channels from the aorta. In order not to complicate the
reconstruction more than was necessary, the entire medial blood
vascular plexus, except the main cardinal tributaries, was omit-
ted. Besides these medial segmental tributaries, two or three
lateral ones develop (figs. 34 and 35) in proximity to the lymph
heart and are closely pressed against the outer side of the myo-
tomes. At a later period, these venules anastomose, become
larger, and combine to form the definitive anterior vertebral vein
and its branches (fig. 35).

86 OTTO F. KAMPMEIER

THE DEVELOPMENT OF THE ANTERIOR LYMPH HEARTS"

Jourdain (’83) probably was the first to make a statement
respecting the development of the lymph hearts in Anura. But
his paper is chiefly concerned with the formation of several lymph
sinuses in the frog, and his allusion to the hearts is very cursory,
these being dismissed in a few sentences. He pointed out that
a small pulsating vesicle, the posterior lymph heart, is visible,
one on each side, at the base of the tail in tadpoles on which the
hind limbs are budding, and that it conducts the lymph into a
branch of the posteardinal vein. On the other hand, the lymph
from the anterior regions of the larva flows directly, according
to him, into the precardinal vein, as in fishes. The anterior pair
of lymph hearts are considered independent (?) structures, which
do not appear until the pectoral girdle has been formed.

13T¢ would seem superfluous again to draw the distinction between ‘lymph
heart’ and ‘lymph sac’ or ‘sinus’, were it not for the confusion of terms and ideas
that is evident in several recent papers on the lymphatic system. In Mrs. Eleanor
L. Clark’s paper (715) on the early lymphatics of the chick, the difference between
lymph sacs and lymph hearts is disregarded, as may be instanced by the follow-
ing quotation: ‘‘According to Baranski and Fedorowicz, the lymph hearts are
formed from two or three lymphatics instead of from a luxuriant plexus, as in
birds and mammals. However, Knower and Kampmeier state that in frog and
toad embryos, the anterior lymph heart is formed from numerous lymphatic
capillaries.’’ In criticism, I wish to state that the researches of Baranski and
Fedorowicz, here mentioned, deal only with the genesis of the posterior lymph
hearts in Anura, and genetic peculiarities distinguish them from their fellows in
the anterior region of the body. Further, Knower is mistated, and my paper,
to which reference was made, published in 1915, is absolutely not concerned with
the development of the anterior pair of lymph hearts, but describes the origin of
a few lymphatic ducts and especially that of the large lymph sinus of the head
which is not the same thing as the lymph heart. My studies of the heart were _
briefly reported for the first time before the American Anatomists during the
Christmas holidays of 1916, a year after Mrs. Clark’s article appeared. In the
Amphibia, lymph heart and lymph sae or sinus are distinct structures, one pos-
sesses muscular walls and pulsates, the other is a modified lymph duct ora trans-
formed lymph capillary plexus.

Miss Sabin also uses lymph heart and lymph sac indiscriminately. Thus
(13, p. 56), she has the following sentence: ‘‘Weliky, Jossifov, and Favaro
thought that the posterior lymph heart arose from the dilatation of the caudal
lymph trunks which grow from the anterior lymph hearts, and Jourdain describes
them as being formed by a rapid destruction of connective tissue.’’ Jourdain’s
account, here mentioned (Comptes Rendues, 96, 1883), does not pertain to the
formation of lymph hearts, but refers to that of the lymph sacs.

DEVELOPMENT OF LYMPHATICS IN ANURA 87

In 1891 Field published his excellent work on the development
of the pronephros in the frog, and in a footnote (91, p. 240)
described briefly, though correctly, a ‘peculiar sac’ which he
found in 8-mm. embryos lateral to the myotomes at the niveau
of the third nephrostome and joined to the postcardinal vein.
‘Respecting the fate and the significance of this singular struc-
ture,” he says, ‘“‘I have no suggestions to offer.” Reference to
this observation is made by Gaupp (’99, pt. 2, p. 380) who ealls
it a ‘Blutblischen’ of unknown function. We now know that
this enigmatical organ is the anterior lymph heart.

Hoyer (05), in his work on the formation of the lymphatic
system in frog larvae, states that the anterior lymph heart makes
its appearance during the stage when the external gills begin to
vanish, as a small spindle-shaped evagination from the short
anterior vertebral vein anlage at the point where this vein
branches dorsally from the pronephric venous plexus. At that
time the walls of the fusiform heart are composed of an inner
endothelium and an outer layer of stellate cells. In 6-mm.
embryos the heart has become larger, but it is still in broad, open
communication with the vein and contains numerous blood cor-
puscles in its cavity. Its walls become thicker and a few cross-
striated muscle fibers are visible in the outer coat. During this
stage in the living specimen the heart occasionally quivers, but
distinct rhythmic contractions do not become evident until later,
when the embryo has reached the length of 12 mm. and the muscle
elements have increased in number and in configuration. In the
meantime valves have appeared, one at the junction of heart and
vein and another at the opening of the lymph vessel into the
heart. After the formation of these structures, blood cells are
only exceptionally found in the heart chamber. The funda-
mental changes in the development of the heart have now occur-
red, and in subsequent stages it merely grows larger and acquires
its definitive character. But it retains its original position
lateral to the second myotome throughout the entire period of
genesis and growth up to metamorphosis.

Knower, in a short paper (08), remarks that the anterior
lymph heart is the first lymphatic to be formed in the frog and

88 OTTO F. KAMPMEIER

agrees with Hoyer that it makes its appearance in approximately
6-mm. embryos (Rana palustris, R. virescens, R. sylvatica).™4
He observed that during this period the heart is situated dorsal
to the posterior end of the pronephros and arises from the 4th
intersegmental vein, thus differing from Hoyer. Knower does
not state explicitly how it originates, but I assume that he regards
it as a local expansion of the vein. According to him, the heart
opens directly into the plexiform venous sinus of the pronephros
just back of the last nephrostome. Striated muscle fibers appear
early in its walls, and in 8-mm. embryos already are arranged in
bundles which branch freely; he believes that these fibers are
derived from the adjacent myotomes, the fourth and the fifth,
since the heart is developed in the intersegment in proximity to
the ventrolateral portions of these muscle segments. Finally, he
notes the development of valves at both the afferent and efferent
portal of the heart.

A very brief preliminary account of the development of the
anterior lymph hearts in Bufo was presented by the writer before
the American Anatomists in 1916.

Morphogenesis

Except that they state definitely the time of appearance and
the location of the anterior lymph heart in frog embryos, neither
Hoyer nor Knower offers a detailed description of its formation;
their accounts are brief and rather general. After more extensive
study, in which numerous graphic reconstructions and some wax
models were made, the writer is able to demonstrate with greater
preciseness, perhaps, its origin and progressive changes. Such
an exposition will show that, in Bufo embryos at least, its genetic
history and the nature of its changes are not so simple as Hoyer’s
and Knower’s descriptions would lead us to suppose. ‘The first
indefinite rudiments are already suggested in approximately
4-mm. embryos (Bufo vulgaris), thus appreciably earlier than

14'The early origin of the anterior lymph heart in the frog was indicated by
Kknower five years earlier (before the American Society of Zoologists, 1903), two
years before Hoyer’s first paper on the development of the lymphatics in the frog
appeared.

DEVELOPMENT OF LYMPHATICS IN ANURA 89

was specified for the frog; yet, | am inclined to believe that even
in frog larvae the first hint of a lymph heart may be demonstrated
earlier by means of reconstructions, which, if accurately executed,

71. ES

ae a 4
cerd.post. = pr oneph. card. post.

Fig. 15 Photomicrograph of a transverse section through the left lateral and
anterior trunk region in a 5-mm. embryo of Bufo vulgaris (IK. E. C., series B 44,
slide 2, section 142). X 340. (Zeiss Apochromat. Obj. 8 and Compensat. Pro-
ject. Oc. 4). 40. seg., 4th intersegmental vein; med. and lat. v. card. post., medial
(subeardinal) and lateral divisions of the posteardinal vein; d. proneph., proneph-
ric or primary excretory duct; ao., aorta; ch., chorda dorsalis; myot., myotome;
ep., epidermis.

bring to view significant twists and turns and other topographical
details that frequently escape the strictest scrutiny of serial sec-
tions. However, the relative time at which the lymph heart
arises is a matter of little importance; we are interested more in

90 OTTO F. KAMPMEIER

the manner in which it originates; but here, too, my observations
are not in agreement with the views expressed by the above-
named investigators. In Bufo embryos at least, it arises neither

_ven-lym.com. jug . et lat. epi

vias
mye?

~*

myot.
> “AE > “3 3 % y

a

v.card, post. ers d.proneph. | ye

Fig. 16 Photomicrograph of a transverse section through the left anterior
lymph heart region in a 5-mm. embryo of Bufo vulgaris (Ik. E. C., series B 44,
slide 2, section 126). > 340. The line of demarcation and the difference of
appearance between the cells of the myotome (myot.) and those of the lymph
heart anlage (cor. lym. ant. sin.) is clearly expressed; ven-lym. com. jug. et lat.,
common segment of the jugular and the lateral line venolymphatics. Other
references as in figure 15.

as an evagination of the anterior vertebral vein nor as a direct
expansion of a particular intersegmental vein, although it is
conceivable how its appearance in certain developmental stages
might lead to such suppositions. The readiest way of obtaining

DEVELOPMENT OF LYMPHATICS IN ANURA 91

a lucid idea of the morphogenesis of the anterior lymph heart is
to examine a consecutive series of reconstructions representing
different genetic stages. Such a series is pictured in figures 31
to 35, inclusive, attention to which has already been directed in
the preceding section on the development of the jugular lymph
duct.

The reconstruction in figure 31, reproducing the conditions in
a 4-mm. embryo, shows the vague beginnings of the anterior
lymph heart as an incipient vascular plexus between the second
and the fourth intersegmental vessels and in connection with the
proximal portion of the third. It is so inconspicuous and ill-
defined that the observer would overlook it but for the striking
changes that occur in the same locality soon after.

In 5-mm. embryos, the above venous, or better, venolymphatic
plexus, the anlage of the anterior lymph heart, has become more
sharply outlined. In comparison with the preceding stage, the
plexus not only has joined the second and fourth intersegmental
vessels by longitudinal anastomosis, but also has increased the
number of its connections with the pronephric sinus from one,
the original mouth of the third intersegmental, to several.

By the distention of the interjoined channels of the lymph-
heart plexus, these coalesce, resulting in a single cavity. In
figure 32 the loop-hole in the anterior part of the anlage is still
indicative of its previous plexiform state. Viewed from the side,
as pictured the contour of the anlage already suggests its future
globular form. In reality, however, its shape at this time is
lenticular, for its lateromedial diameter is not much greater than
the third intersegmental vessel from which it sprang, and accord-
ingly in transverse section through its center (fig. 17, cor. lym.
ant. sin.) it would appear as a spindle-shaped expansion of this
vessel. The connection of the heart with the pronephric venous
sinus and with the surrounding intersegmental network, which,
as already shown, is involved in the formation of lymphatic ducts,
vary little in position and in number, as a comparison of several
specimens of the same age has shown. At the lower margin of
the lymph-heart anlage (fig. 32) the delta-like confluence with
the pronephric sinusoids is to be regarded as a complication of the

92 OTTO F. KAMPMEIER

mouth of the former third intersegmental vein, and the extensions
at the upper margin as the distal portion of this vessel. The
anterior and posterior junctions of the lymph heart with the

“ven-lym. com. jug. et

¥:. «6S ?
> oa

nS ee:
. proneph

Fig. 17 Photomicrograph of a transverse section through the left anterior
lymph heart region in a 6-mm. embryo of Bufo vulgaris (IX. E. C., series B 54,
slide 2, section 130). X 340. si. proneph., the mouth of the original 3rd inter-
segmental vein, branch of the pronephric sinus; proneph., pronephric tubule;
med. spin., medulla spinalis. Other references as in figure 15.

second and fourth intersegmentals, respectively, may also be
resolved into plexiform channels. Thus, four fairly constant
groups of connections may be recognized, a ventral, a dorsal,
an anterior, and a posterior group.

DEVELOPMENT OF LYMPHATICS IN ANURA 93

In 7-mm. embryos, the connections between the lymph heart
and the cireumjacent venolymphatic plexus begin to break away.
An early stage in this process is shown in figure 33. The anterior
connection is still broad; on the dorsal surface of the heart one
has already severed relations and another is very much con-
stricted; the one on the posterior surface, too, shows signs of
contraction when compared with its homologue in figure 32. On
the ventral aspect of the heart one channel of confluence is just
being pinched off, but the more anterior connections are fusing
into one and so constitute the anlage of the anterior vertebral
vein and the lymphaticovenous tap. During these progressive
events, the heart becomes more spheroidal as the area between
the myotomes and the epidermis widens, associated with the
rounding out of the back and sides of the embryo (fig. 18).

In 8-mm. embryos, mere vestiges, in the form of small pro-
jections, remain of the former union between the lymph heart and
the neighboring lymph vessels, as delineated in the reconstruction
in figure 35, but in every case their coincidence with the points
of union of earlier stages can be made out readily. At this period,
then, there seems to be no open passage whatsoever between the
cavity of the lymph heart and the remainder of the lymphatic
conduit system. It is a blind globular chamber attached to the
anlage of the vertebral vein at its anterior and ventromedial
surface and is confluent with it.

During the period between 8- and 10-mm. stages the secondary
or permanent communication is established between the lymph
heart and the afferent lymph duct. A comparison of figures 34
and 35 and the photomicrographs, figures 19 to 28, shows plainly
how this is accomplished. By uniform growth and dilatation of
the lymph heart as well as of the cireumjacent lymph vessels,
the common segment of the jugular and lateral ducts (lym. com.
jug. et. lat.) and the dorsal aspect of the heart are gradually
brought together, this approximation continuing until the duct
comes to lie in a shallow groove-like indentation or depression of
the heart wall. Along this line of contact the first afferent
ostium appears. In later stages, as more tributaries of the afore-
said lymph channels are formed, some of them, situated nearest
the heart, cross over its surface and come to lie against it, and

94. OTTO F. KAMPMEIER

eventually break through at certain points, so increasing the num-
ber of portals of entry for the lymph stream, as shown in the
drawing (fig. 24) of the lymph heart ina young toad. In figure 35

HP lym.com. juget lot. jee aM ep

AL

si.proneph. ||

Fig. 18 Photomicrograph of a transverse section through the left anterior
lymph heart region in a 7-mm. embryo of Bufo vulgaris (K. E. C., series B 27,
slide.2, section 83). X 340. lym. com. jug. et lat., common segment of the
lymphatica jugularis and lymphatica lateralis; *, temporary breaking away of
the lymphatics just mentioned from the lymph heart (cor. lym. ant. sin.). Other
references as in the preceding figure.

such a condition is already intimated by the lymph vessel which
branches off from the jugular duct and passes diagonally over the
outer surface of the lymph heart.!

' During the period of metamorphosis, the plexus of lymphatic vessels in the
vicinity of the anterior lymph heart develop into the sub-scapular lymph sinus.
The description of the transformation of the lymphatie system in the tadpole

DEVELOPMENT OF LYMPHATICS IN ANURA 95

One detail still remains to be considered in the morphogenesis of
the anterior lymph heart, namely, the shifting of the efferent portal,
or ostium venosum. In the earlier stages, this connection is with

Cu eo ee NR se) Ss

lym. com. jwg.et lat.

my |

et

X
XK

ines

Fig. 19 Photomicrograph of a transverse section through the left anterior
lymph heart region in an 8-mm. embryo of Bufo vulgaris (K. E. C., series B 49,
slide 2, section 112). X 340. v. vert. ant., vena vertebralis anterior, branch of
the pronephric sinus (si. proneph.), its mouth being that of the original third
intersegmental vein; *, point of former connection, in the form of a small spur,
between the common segment of the jugular and lateral lymphatics and the
lymph heart; gan. I//, third spinal ganglion. Other references as before.

the pronephric venous sinus and is found squarely on the ventral
side of the heart (fig. 32). This condition is changed by three
factors: first, the breaking away of the posterior channel of the

into that of the fully formed animal represents another section of the original
monograph and will appear later as a separate paper.

96 OTTO F. KAMPMEIER

multiple junction and the amalgamation of the other channels
into a larger one: secondly, the outgrowth of the anterior verte-
bral vein (v. vert. ant.) just internal to the latter, and, thirdly,
the distention of the lymph-heart cavity, the bulging of which
is more pronounced laterally than medially where the myotomes
resist its expansion (ef. photomicrographs figs. 18 to 22). Asa
result of the interaction of these factors, the lymphaticovenous
.tap is shifted forward and medially. The ultimate condition has
not yet been attained, however, for in the young toad the junction
is at the anterior, more conical, end of the heart (fig. 24). Be-
tween the stage figured in figure 35 and the final one, it is evident,
therefore, that considerable displacement still occurs, but to
specify all of the underlying causes is impossible and is of little
importance. Unquestionably, it is correlated with the stresses
and strains due to other bodily changes that take place in the
neighborhood of the lymph heart during development, such as
the atrophy of the pronephros and the proximal segment of the
posteardinal,'® the absorption of the pronephric sinus by the
internal jugular, the consequent shifting of the mouth of the
anterior vertebral vein, the differentiation of the myotomes, and
the rearrangement of the resulting muscles, to mention only a few
of the most evident modifications.

Histogenesis

a. The lymph heart wall. Waving described the conformation
of the lymph heart and its venous relations, the development of
its walls and valves remains to be discussed.

In early stages (4- and 5-mm. embryos) when the anterior
intersegmental vessels have just been established, the area
between the epidermis and the myotomes is very narrow, not
much wider than is sufficient to accommodate these vessels
(fig. 15). The mesenchyme, too, is very scanty here except in
the region of the 3rd intersegmental vein, where its yolk-laden
cells soon become more numerous and are locally massed against

16'The medial division of the postcardinal vein has been shown by the writer
(Anat. Rec., vol. 19, 1920) to correspond to the sub-cardinal vein of higher verte-
brates.

DEVELOPMENT OF LYMPHATICS IN ANURA 97

the confines of that channel. This is especially true during the
stage when the lymph-heart anlage is plexiform, a section of
which is shown in the photomicrograph, figure 16 (cor. lym. ant.
sin.). As suggested in the figure, the masses of mesenchymal
cells are not uniformly arranged around the outlines of the lymph-
heart plexus, but are irregularly distributed, at one level being
crowded against its lateral side, at another, against its medial,
and more frequently between the meshes of its interanastomosing
channels. Incidentally, it is evident that the reconstruction,
which simply represents an enlarged cast of the lumen of the
channels, does not exhibit all of the essential features of the
developing structure, and to acquire a correct conception of the
genetic processes, the sections as portrayed in the photomicro-
graphs must be examined together with the reconstructions.
The mesenchymal cells are so closely packed together and so
filled with yolk globules that it is impossible to determine their
individual boundaries. At several points, too, such aggregations
seem to bound the cavity of the lymph heart anlage directly,
at least no distinct intima lining it can be recognized. Otherwise
the lining is quite sharply defined, though only part of the endo-
thelial cells tend to the flattened shape, while others still retain
the unspecialized form in which the nuclei are in general either
oval or spherical and resemble those of ordinary mesenchymal
cells. The nuclei of these mesenchymal masses stain deeply and
are coarsely chromatic, and many of them have an indented
circumference which conforms to the large yolk bodies in the
cytoplasm.

At another level of the lymph-heart plexus, Just back of the
section shown in figure 16, primitive spherical blood cells, also
stuffed with yolk globules, are crowded together and block the
lumen of a connecting channel. Generally speaking, there is
already a marked difference between the nuclei of circulating
blood cells and those of mesenchymal cells; the former are dense
and opaque and take almost a black color when stained with
haemotoxylin, while the latter possess lighter staining areas
between the large chromatic granules. A few exceptions, how-
ever, were observed; several of the nuclei of the mesenchymal

QS OTTO F. KAMPMEIER

cell aggregations approach the haemal nuclei in density,though
the writer is unable to demonstrate decisively the transition and
conversion of one into the other. This observation immediately
calls to mind the researches of Miller (713) and Allen (’13) on the
development of the thoracic duct in the chick and the caudal
lymph heart in Polistotrema stouti, respectively, where it was
discovered that some mesenchymal cells were converted into
blood cells during the early genetic stages of these lymphatics.
Consequently the question arises: Does the incipient anterior
lymph heart in Anura also function transiently as a haemopoietic
organ? Do some of the cells of the mesenchymal masses contig-
uous to and between the channels of the plexiform anlage become
differentiated into blood corpuscles? All my efforts to demon-
strate this proved futile in the face of that prime obstacle, the
abundance of yolk, which obscures and erases the more delicate
tissue distinctions.

In 6-mm. embryos not only has the periphery of the lymph-
heart lumen become more definite than in the previous stage,
but the surrounding masses of mesenchymal cells are becoming
more evenly spread out over its outer surface (fig. 17).

In the next older stage (7 mm.) the rearrangement of the cells
composing the walls of the lymph heart is such that in general
two layers may be distinguished (fig. 18), a lining or internal
layer and a covering layer, but which, as yet, are not sharply
delimited. This indistinctness is further emphasized by the fact
that the intimal cells are not all flattened, as we should expect
in this relatively advanced stage, but still retain their general-
ized character. Indeed, one is not able to discern any striking
difference between them and the other mesenchymal cells; as
regards size, form, and appearance, the nuclei seem identical.
The yolk globules have decreased in number in both layers, and
for the first time one can get a glimpse of the shape of the cell
body. Some of the cells of the outer or covering layer are
becoming definitely fusiform, with their long axis directed
parallel to the circumference of the heart cavity. A considerable
number of blood cells are present in the latter (figs. 18 to 22),
a fact of no special significance, however, for the heart is in broad

DEVELOPMENT OF LYMPHATICS IN ANURA 99

open communication with the pronephric venous sinus and no
valve has yet been established at this lymphaticovenous junction.

In the subsequent period of development the wall of the lymph
heart changes very slowly in character. Instead of increasing
in thickness, it becomes relatively thinner during the time be-
tween 9- and 13- or 14-mm. embryos, due, in the first place, to
the progressive flattening of the cells of the intima layer; secondly,
to the attenuation of the covering cells, and, thirdly, to the loss
of the large yolk corpuscles. In fact, during these stages, the
second or covering layer does not form a complete investment of
the endothelium, for there are bare spots (figs. 20 and 21) where
endothelium constitutes the only line of demarcation between
the cavity of the lymph heart and the surrounding mesenchymal
reticulum. ‘The scantiness of the covering layer at this time is
probably explained by the slow specialization of its cells and the
more rapid expansion of the heart lumen with the resultant
stretching of its lining cells. In 16-mm. embryos it again forms a
continuous single cell-sheet (fig. 23), though it is still quite as
thin as the endothelial layer, and is composed of slender spindle-
shaped cells which show delicate striations. These cells, differ-
entiated, as we have seen, from the mesenchymal cell aggrega-
tions so conspicuous during the initial stages of the lymph heart,
compose the anlage of its muscle coat.

Knower claims there is evidence that the cells of the muscle
coat are derived from the adjacent myotomes, but the writer is
unable to furnish proof for the contention. In 5-mm. embryos
a radical difference already obtains between the cells of the
myotomes and those which surround the lymph-heart anlage—a
distinction strikingly revealed in figure 16. Nevertheless, this
fact does not discount the possibility that in much earlier stages
the potential muscle cells of the lymph heart may proliferate
from the myotomal elements; but, if this is found to be true, then
it will be equally true that other mesenchymal cells of the same
region have a similar source, provided the absence of any visible
difference whatsoever between the cells of the mesenchyme and
those of the lymph-heart anlage is any criterion of the similarity
of origin.

100 OTTO F. KAMPMEIER

The further development and thickening of the muscle coat
is not consummated until some time after metamorphosis, for
even in the young toad it is not conspicuous and is composed of
only three or four cell layers.

; <j Se aa we EE eee

lym. com. jug: ef lat.

_ \ym, lat. Bee

Fig. 20 Same, in a 9-mm. embryo of Bufo vulgaris (K. E. C., series B 13,
slide 3, section 14). 340. *, the common segment of the jugular and lateral
lymphatics by expansion have again come in contact with the lymph heart;
lym. lat., a ventral branch of the lateral lymphatic (lymphatica lateralis) and con-
tinuous with the dorsal one at a further level. Other references as before.

The intima, too, of the lymph heart is slow in acquiring its
definitive character, which is not attained until approximately in
15- or 16-mm. stages. Even in 10- or 11l-mm. embryos the
endothelial cells show little advance over those present in the
heart of somewhat younger individuals. Some do have the finished

~ \ym.,
gs

tee “lym.com.ju - et. lat.

Fig. 21 Photomicrograph of a transverse section through the left anterior
lymph heart region in a 10-mm. embryo of Bufo vulgaris (K. E. C., series B 34,
slide 2, section 83). > 340. *, the common segment of the jugular and lateral
lymphatics has indented the dorsal wall of the lymph heart, which has become
thickened at this point. Other references as before.

Fig. 22 Same, in a 12-mm. embryo of Bufo vulgaris (Kk. E. C., series B 11,
slide 3, section 34). X 340. *, the wall between the lymph duct and the lymph
heart has broken through in the middle and the two flaps so formed represent the
valves of the afferent portal. Other references as before.

101

102 OTTO F. KAMPMEIER

form, in so far as their nuclei appear compressed and uniformly
dense, but others again contain large, spherical, and coarsely
chromatic nuclei and protrude into the heart cavity like little
humps or hillocks, which call to mind the observations of certain
investigators on haemopoiesis where endothelium germinated
blood cells, but the author was unable to discover an undoubted
case where one became constricted off.

The further differentiation and thickening of the heart wall
occurs during the period of growth after metamorphosis, and
histological examination of a section through the lymph heart of
the adult anuran reveals three well-defined coats or layers: a
tunica interna or intima, a tunica media, and a tunica externa
or adventitia. The first is composed of the layer of highly
flattened lining cells and a very thin stratum of connective tissue,
probably elastic in nature, immediately external to them. As we
should expect from the great energy displayed by the lymph
hearts during life, the muscular tunica media, the second coat,
is the broadest layer of the heart. Its muscle cells or fibers are
of varying length and thickness and group themselves into small
bundles which branch and interlace in a complex manner. Hoyer
(704) claims that the individual muscle fibers themselves branch
and anastomose and possess numerous cross bands, which call to
mind the intercalated dises of human cardiac muscle. A large
number of elastic strands are also contained in the media. No
sharp boundaries exist between media and adventitia. ‘The
latter is made up of fibrillar connective tissue in which are seat-
tered pigment cells. The nerve fibers to the anterior pair of
lymph hearts are apparently supplied by the III spinal nerve.
According to Waldeyer (’64), both medullated and non-medul-
lated nerve fibers are found in the walls of the fully developed
lymph hearts.

Before discussing the formation of the valves, a variable feature
may be mentioned in connection with the development of the
walls. In about half of the lymph hearts examined between 8-
and 16-mm. stages, a strand or trabecula, sometimes delicate
and sometimes fairly thick, bridged the cavity. Occasionally
they were imperfect, simply projecting as slender filaments

DEVELOPMENT OF LYMPHATICS IN ANURA 103

(fig. 26). It is possible that these trabeculae correspond to the
incomplete partition which, according to Radwanska (’06), is of
constant occurrence in the anterior lymph hearts of adult frogs.

b. The afferent portals. As was indicated earlier, during the
first part of its functional life the lymph heart of the anuran
embryo possesses but two valvular openings, a lymphaticovenous
or efferent one and the entrance of the afferent lymph vessel.
It is only in later embryonic and postmetamorphic periods that
the number of afferent gateways is increased from one to about
twelve. The development of this type will be considered first.

In the discussion of the morphogenesis of the lymph heart the
writer has described how the developing lymphatic plexus sur-
rounding it temporarily detaches and recedes from it and how
the longitudinal channel of the plexus, the common segment of
the jugular and lateral-line ducts lying dorsal to the heart, again
comes into juxtaposition with it by the dilatation of both struc-
tures, whereupon the permanent communication is established.
Figure 20 is a section of the lymph-heart region during the phase
of simple apposition. Here the heart wall, having the same
appearance and thickness as elsewhere along its periphery, sepa-
rates the cavity of the heart (cor. lym. ant. sin.) from that of the
lymph duct (lym. com. jug. et lat.), and there is as yet no indica-
tion of the future opening between the two. In the next older
stage (10-mm.), the heart and vessel are more intimately applied
to each other by the partial invagination of the latter into the
heart, as shown in figure 21 (*); in the reconstruction (fig. 35) the
vessel lies in a shallow furrow of its heart wall at af. It is along
this surface of contact that the partition dividing the two cavities
thickens considerably (fig. 21) by the proliferation of its cells:
Somewhat later, a cleft develops in the center of the thickened
area (fig. 22, *) by the separation of its cells, evidently the effect
of the increasing pressure within the afferent lymph duct. The
margins of the simple rupture now serve as the valve. These,
by further proliferation, may become longer, and as they converge
and project into the lumen of the lymph heart they produce the
typical teat-like form in section (fig. 23, *). The other valvular
afferent portals which arise later (fig. 24, af.) are developed in

104. OTTO F. KAMPMEIER

a similar way. In a young toad (Bufo lentiginosus), shortly
after the period of metamorphosis, the author observed five such
points of entry in the anterior lymph heart, but doubtlessly their

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ak tee

Fig. 23 Photomicrograph of a transverse section through the left anterior
lymph heart region in a 16-mm. embryo of Bufo vulgaris (KX. E. C., series B 39,
slide 5, section 51). X 340. *, valve at the afferent portal. Other references
as before.

number is increased with the growth of the toad towards maturity,
for Radwanska (’06) counted more than a dozen on the same
organs in adult frogs.

c. The efferent portal, or ostiwm venosum. ‘The formation of
the valve at the lymphaticovenous junction is perhaps not so

DEVELOPMENT OF LYMPHATICS IN ANURA 105

diagrammatic. It develops, however, at the same time as the
other valve. In 7-mm. embryos the lymph heart is still in broad
open communication with the anlage of the vertebral vein (fig. 18).
In the next few succeeding stages (8-, 9-, and 10-mm. embryos)
the junction becomes progressively constricted by the local
thickening of its surrounding wall. In fact, in some cases it
was observed that the cell proliferation was so considerable as to
block almost entirely the channel of connection (figs. 19 and 22).

cor lym. ant.

Si. SUDSCAD.

Fig. 24 Quasischematic reconstruction of the left anterior lymph heart of
the young toad immediately after metamorphosis. 150. Ventromedial
view. cor lym. ant., cor lymphaticum anterius; v. vert. ant., vena vertebralis
anterior; si. subscap., subscapular sinus; af., one of the afferent portals.

Then, by the elongation of its thickened sides (fig. 25,*) asso-
ciated with the expansion of the venous lumen up and around it
towards the lymph-heart wall, the connection becomes telescoped,
as it were, into the cavity of the vein, so that the thickened cell
masses project as the lips of the valve (fig. 26,*). This process
is completed in 10- to 12-mm. toad embryos (B. vulgaris). In
the outline sketches in figure 27, the formation of both the afferent
and the efferent portal is expressed graphically.

v.vert. ant.

Ds

§ cor lym.ant. sin. {§§ lym. tat.

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yes rm : .¢@

”

.

Fig. 25 Photomicrograph of a transverse section through the left anterior
lymph heart region in a 10-mm. embryo of Bufo vulgaris (IX. E. C., series B 34,
slide 2, section 68). X 340. *, formation of the valve at the efferent portal.

* Other references as before.

Fig. 26 Same, in a 12-mm. embryo of Bufo vulgaris (K. E. C., series B 11,

slide 3, section 41). X 340. *, valve of the efferent portal. Other references

as before.
106

DEVELOPMENT OF LYMPHATICS IN ANURA 107

Fig. 27 Diagrams (1 to 7) illustrating the formation of the afferent and effer-
ent ostia of the lymph heart (based on transverse sections). v., vein; h., lymph
heart; v. l., venolymphatic, a channel of the intersegmental vein plexus, and
converted into the afferent lymph vessel, J.

108 OTTO F. KAMPMEIER

Since blood cells have free access to the cavity of the lymph
heart before the appearance of the valve at the lymphatico-
venous junction, in stages up to and including 10-mm. embryos,
they are abundant in it (figs. 18 to 22 and 25). In 12-mm.
embryos and later (figs. 23 and 26), they are rarely present, and
we may conclude from this and the fact that the valves are now
functionally complete and efficient that the pulsations of the
lymph heart commence at this time, for the first few contractions
would certainly cause the evacuation of all haemal elements.

On account of the abundance of pigment in the integument of
Bufo embryos, it was impossible to determine accurately by direct
observation on the living specimen at which time the pulsations
of the anterior lymph heart commenced, but, according to Hoyer
(05), they first become evident as irregular quiverings in the
more transparent frog embryos (R. temporaria) when they are
12 to 18 mm. long. Later the pulsations of the lymphatic heart
become more rhythmic, but the beats coincide neither with those of
the haemal heart nor with those of its companion on the opposite
side. In the mature animal it throbs as often as sixty to seventy
times every minute, and since its capacity is about 0.5 cu. mm.
(Radwanska, ’06), the quantity of lymph pumped into the
anterior vertebral vein during this period is 30 cu. mm., and in
one hour reaches the relatively considerable amount of 180 cu.
mm. During systole of the lymph heart, the efferent valve,
projecting into the vein, opens for the discharge of the lymph,
but closes and prevents the backflow of blood into the heart
chamber during diastole. Similarly, the afferent gateways per-
mit the entrance of the lymphatic current from the cireumjacent
lymph sinuses, yet avert its reflux during systole.

DEVELOPMENT OF LYMPHATICS IN ANURA 109

SUMMARY

1. On the development of the primary maxillary lymph sinus

The sinus begins in approximately 5-mm. embryos of Bufo
vulgaris in the form of small discontinuous anlagen, which appear
either as cellular thickenings of the endothelium of the develop-
ing jugular veins or as islands lying in the mesenchyma in the
immediate vicinity of these vessels.

During development all vascular anlagen of the head region,
both haemal and lymphatic, can be distinguished from the sur-
rounding mesenchyma by the greater number of yolk globules
present in their endothelium.

The originally solid lymphatic anlagen acquire lumina, which
have their inception as small crevice-like spaces in the cytoplasm
between the large yolk globules.

By continued proliferation and growth, the individual anlagen
increase in length, bud collateral branches, coalesce with one
another, and in time form a complex tubular network extending
in a curved plane from the region of one external jugular vein to
that of the opposite side; this network represents the principal
or mandibular division of the primary maxillary lymph sinus.

The other divisions, the circumoral, temporal, and pericardial,
arise from the mandibular division by outgrowth and extension.

The lymphatic network becomes transformed into a spacious
and uninterrupted sinus by the progressive expansion of all the
anastomosing channels and by the reduction and tearing of the
intervening mesenchymal strands and trabeculae.

During the preceding genetic stages, the sinus possesses no
outlet; it is not confluent with the veins. The sinus receives an
outlet in approximately 10-mm. embryos as the posterior pro-
longations of its temporal divisions join the jugular lymphatics
and thereby are placed in communication with the anterior lymph
hearts and through them with the venous system.

The extension and distention of the developing sinus are prob-
ably achieved by the increasing internal pressure on its walls of
the accumulating lymph before an exit is established. Duringthe
expansion of the sinus, the lining cells become progressively
flattened and assume typical endothelial qualities.

110 OTTO F. KAMPMEIER

2. On the development of the jugular lymphatic

In 5- to 6-mm. embryos, the first three intersegmental veins,
which are dorsal vertical tributaries of the pronephric venous
sinus (common segment of pre- and postcardinal veins), become
joined longitudinally by imteranastomoses and consequently
take on a plexiform character.

The aforesaid intersegmental vein plexus, which in view of its
original relations and its future function may be called a veno-
lymphatic one, gives rise to the jugular lymphatic, the important
change consisting in its gradual separation from the veins (pro-
nephric venous sinus).

By the expansion, approximation, and fusion of the longi-
tudinal components of the plexus, the main channel of the jugular
lymphatic is definitely established, and it eventually makes con-
nection -anteriorly with the temporal division of the primary
maxillary lymph sinus and at its posterior end, in common with
the lateral line lymphatic, joins the anterior lymph heart.

3. On the development of the anterior lymph heart

The anterior lymph heart, on either side, arises from a cir-
cumscribed portion of the venolymphatie plexus, mentioned in
the preceding section, at the level and in the axis of the original
3rd intersegmental vein.

The plexiform anlage of the lymph heart becomes transformed
into the uninterrupted heart chamber by the expansion and fusion
of its interjoied channels.

The developing lymph heart in approximately 7- or 8-mm.
embryos severs connection with the cireumjacent venolymphatic
plexus, but remains in continuity with the venous system via
the mouth of the former 3rd intersegmental vein, now the mouth
of the anterior vertebral vein.

A communication is reestablished between lymph heart and
afferent lymphatic, the common segment of the jugular and
lateral-line lymphatics, in approximately 10-mm. embryos; this
is accomplished by the gradual approximation of the two struc-
tures, due to their growth and dilatation, and by the perforation

DEVELOPMENT OF LYMPHATICS IN ANURA tel

of the intervening wall at the line of contact to form a teat-like
valve. Other permanent afferent portals are formed later in a
similar manner, there being five of these in Bufo lentiginosus
immediately after the period of metamorphosis.

The valve at the efferent or lymphaticovenous tap is developed
from a circular endothelial cushion projecting into the lumen of
the anterior vertebral vein, followed by the telescoping of this
valvular portion of the heart deeper into the lumen of the vein.

During the later development of the heart the efferent tap is
shifted forward from a ventral position on the heart to an ante-
rior one.

While the heart is expanding, the lining cells become pro-
gressively flattened; the mesenchymal cells external to these
become spindle shaped and ultimately develop into muscle cells.

Before the efferent valve has become differentiated, numerous
blood corpuscles are found in the heart cavity. The evacuation
of these elements doubtlessly occurs at the first vigorous con-
tractions of the heart.

LITERATURE CITED

Auten, W. F. 1913 Studies on the development of the veno-lymphatics in the
tail region of Polistotrema (Bdellostoma) stouti. First communica-
tion: Formation of the caudal hearts. Quart. Jour. Microsc. Sci.,
vol. 59, part 2, July.

Cuark, Exeanor L. 1915 Observations of the lymph flow and associated
morphological changes in the early superficial lymphatics of chick
embryos. Am. Jour. Anat., vol. 18, November.

Fretp, H. H. 1891 The development of the pronephros and segmental duct
in Amphibia. Bull. Museum Comp. Zool., vol. 21.

Gaupp, E. 1899 Ecker und Wiederscheim’s Anatomie des Frosches (Zweite
Abteilung). Braunschweig.

Hoyer, H. 1904 Uber die Lymphherzen der Frésche. Bull. Acad. d. Sc. de
Cracovie, Mai.

1905 Uber das Lymphsystem der Froschlarven. Anat. Anz., Bd. 27.
Ergiinz. Heft, 1905, und Bull. Acad. d. Se. de Cracovie, Juli, 1905.
1908 Untersuchungen iiber das Lymphgefisssystem der Froschlarven.
Zweiter Teil, Bull. Acad. d. Se. de Cracovie, Mai.

Huntineton, Gro. 8. 1911 The development of the lymphatic system in rep-
tiles. Anat. Rec., vol. 5.

Jourpatn, L. 1883 Sur le systeme lymphatique des tetards et grenouilles.
Comptes Rendus d. l’Acad. d. Se., T. 96.

1 el 24 OTTO F. KAMPMEIER

IXAMPMEIER, Ortro F. 1912 The value of the injection method in the study of
lymphatie development. Anat. Ree., vol. 6.

1912 The development of the thoracie duct in the pig. Am. Jour.
Anat., vol. 13, September.

1915 On the origin of lymphatics in Bufo. Am. Jour. Anat., vol. 17,
January.

1917 The formation of the anterior lymph hearts and neighboring
lymph channels in Bufo. Abstract of preliminary Report. Anat.
Ree., vol. 11, January. Read before Am. Anatomists, 33rd Session,
New York, Christmas, 1916.

1919 A summary of a monograph on the morphology of the lymphatic
system in the anuran Amphibia, with especial reference to its origin
and development. Anat. Rec., vol. 16, August.

1920 The changes of the systemic venous plan during development and
the relation of the lymph hearts to them in Anura. Anat. Rec., vol.
19, July.

Knower, H. McE. 1908 The origin and development of the anterior lymph
hearts and the subcutaneous lymph sacs in the frog. Anat. Ree., vol.
2. Proceed. Am. Assoc. Anat., 24th Session.

McCuure, C. F. W. 1915 The development of the lymphatic system in fishes
with especial reference to its development in the trout. Memoir
no. 4, Wistar Institute of Anatomy, Philadelphia. Preliminary Report
in Anat. Ree., vol. 8, 1914.

Miututer, A. M. 1913 Histogenesis and morphogenesis of the thoracic duct
in the chick; development of blood cells and their passage to the blood
stream via the thoracic duct. Am. Jour. Anat., vol. 15.

RapwanskKA, Marie 1906 Die vorderen Lymphherzen des Frosches. Bull.
Acad. Se. de Cracovie, Mai.

SABIN, FLORENCE R. 1913 The origin and development of the lymphatic sys-
tem. Johns Hopkins Hospital Reports. Monographs, new series,

no. 5.
WieperscHerm, R. 1909 Vergleichende Anatomie der Wirbeltiere; 7te Auflage.
Jena.
- APPENDIX

At the time when the above paper had already appeared in proof, I found a
reference in the literature to an article by Bles on “‘The life-history of Xenopus
laevis’? (Trans. Roy. Soc. Edinb., vol. xli, 1905) in which he described and pic-
tured the anterior lymph hearts in the larvae of this anuran. Reference to this
paper will be made in my work on the comparative morphology of the systemic
lymphatics which is in preparation.

PLATE 1
EXPLANATION OF FIGURE

28 Reconstruction of the larger haemal and lymphatic vessels in the head
and anterior trunk region of a 7.5-mm. embryo of Bufo lentiginosus (K. E. C.,
series B31), dorsal view. X 50.

Structures not colored: lymphatics, anterior end of spinal cord and brain,
olfactory, optic, and auditory vesicles, and pronephros and its duct; the latter
structures omitted on the right side.

Lymphaties: si. circ. or., cireumoral division of the sinus maxillaris primi-
genius; st. mand., mandibular division; sz. temp., temporal division; sz. pericard.,
pericardial division; lym. jug., lymphatica jugularis; cor. lym. ant. dex. and sin.,
cor lymphaticum anterius dextrum and sinistrum; lym. lat., lymphatica lateralis.

Veins (blue): A portion of the sinus venosus is shown ventral to the myelen-
cephalon joined by the hepatic sinusoids, the external jugular veins and cuvierian
ducts. The external jugular accompanies the pericardial lymphatic and anteri-
orly receives two branches, a medial (hidden by the mesencephalon), probably
the anlage of the vena lingualis, and a lateral, lying closely against the inner side
of the principal and cireumoral divisions of the primary maxillary sinus and repre-
senting the future vena mandibularis and branches. In the region of the prone-
phriec sinusoids (a large section omitted on the right side) the cuvierian duct is
joined by the precardinal or internal jugular, which passes laterally around the
auditory vesicle and possesses three large tributaries, the vena orbitonasalis,
the vena ophthalmica, and a large intracranial vein. The lateral and medial
(subeardinal) divisions of the posteardinal, situated along the pronephric duct,
and anteriorly, near the pronephric sinus, receive the anterior vertebral vein
into which the anterior lymph heart opens.

Arteries (red): The heart, external carotids and ventral roots of the aortic
arches are not shown in the drawing. The radices aortae are broadly divergent
in the region of the auditory vesicles, where they connect with the dorsal roots of
the aortic arches which, as they curve ventrad, lie closely against the inner side
of the temporal lymphatics. Anteriorly the radices aortae are continued forward
as the internal carotids which give off in the order named the following impor- -
tant branches: arteria palatina, a. ophthalmica, and a. carotis cerebralis. The
pronephric glomeruli branch from the radices aortae immediately anterior to their
convergence and fusion to form one trunk (ventral to the spinal cord).

114

DEVELOPMENT OF LYMPHATICS IN ANURA
‘0 F. KAMPMEIER

THE AMERICAN JOURNAL OF A

PLATE 2
EXPLANATION OF FIGURE

29 Reconstruction of the vascular channels of the ventral cephalic region in
a 6-mm. embryo of Bufo vulgaris (IK. E. C., series B 53), ventrai veiw. X 125.

v. jug. ext., vena jugularis externa.

a. car. ext., arteria carotis externa.

g. thyr., glandula thyroidea.

ao., aortic arches.

vent., ventriculus of the heart.

d. Cuv., ductus Cuvieri.

sin. ven., Sinus venosus; its cut edge shows its attachment to the liver, for at
this period the hepatic sinusoids open directly into it.

The lymphatics, the anlagen of the sinus maxillaris primigenius arise inde-
pendently of each other along the venous components of the jugulocarotid
plexus; some of them have severed contact with the blood vessel wall, while
others still adhere to it.

DEVELOPMENT OF LYMPHATICS I N PLATE 2
OTTO F. KAMPMEIER

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PLATE 4
EXPLANATION OF FIGURE

31 Reconstruction of the vascular channels and other structures in the
region of the left pronephros in a 4-mm. embryo of Bufo vulgaris (K. E. C., series
B 45), lateral view. X 166.

1-4 v. seg., 1st to the 4th intersegmental veins; the beginning of the formation
of the venous plexus in the development of the anterior lymph heart is already
indicated by the short and irregular branches of the 3rd intersegmental vein.

. jug. wnt., vena jugularis interna (preecardinal)
. Jug. ext. vena jugularis externa
. card. post., vena cardinalis externa

d. Cuv., ductus Cuvieri

nephst., I, II, et IIT, nephrostomes of the pronephros

d. proneph., pronephric or primary excretory duct

med. spin.,- medulla spinalis

IT, I1T, IV, 2nd, 3rd and 4th spinal ganglia

vag., a ganglionic prolongation from the vagus group back along the medulla
oblongata; it may be a vestige associated with the lateral-line organs and later
disappears.

A reconstruction of the above structures in a 5-mm. embryo, representing an
intermediate stage between that pictured on the opposite plate and that on the
following one, was omitted with several other illustrations to reduce the cost of
publication, although it revealed very strikingly the lymph heart plexus before
its coalescence into a uniform cavity, a process almost completed in plate 5.

see

122

DEVELOPMENT OF LYMPHATICS I PLATE 4
TO F. KAMPMEIER

PLATE 5

EXPLANATION OF FIGURE

32 Reconstruction of the vascular channels and other structures in the region
of the left pronephros in a 6-mm. embryo of Bufo vulgaris (K. E. C., series B 54),
lateral view. X 166.

1-4 v. seg., Ist to the 4th intersegmental veins; by the formation of interan-
astomosis between them a plexus results, which may be called a venolymphatic
one, in view of the fact that it subsequently gives rise to the lymph vessels in this
region.

cor. lym. ant., anlage of the anterior lymph heart.

. Jug. int., vena jugularis interna.

. Jug. ext., vena jugularis externa.

. card. post., vena cardinalis posterior.
. Cuv., duetus Cuvieri.

d.

proneph., pronephrie duct.

nephst. I, II, et IIT, pronephrie nephrostomes.

med. spin., medulla spinalis.

I. II, III and IV, spinal ganglia; the 1st spinal ganglion is a very transitory
and vestigial structure appearing for the first time in 6-mm. embryos and disap-
pearing very soon after; the 2nd spinal ganglion becomes the Ist of the adult.

vag., ganglionic prolongation of the vagus group (ef. fig. 31).

124

DEVELOPMENT OF LYMPHATICS IN ANU PLATE 5
OTTO F. KAMPME‘ER

med. spin.

cor lym.ant.
nephst.1 etm

PLATE 6
EXPLANATION OF FIGURE

33. Reconstruction of the vascular channels and other structures in the
region of the left pronephros in a 7-mm. embryo of Bufo vulgaris (IX. E. C., series
B 27), lateral view. X 166.

lym. jug., lymphatica jugularis; at the points marked * there is still a minute
connection between the pronephric sinus and the lymphatic.

temp. s. max. prim., posterior tip of the temporal division of the sinus maxil-
laris primigenius.

lym. lat., lymphatica lateralis; this lymphatic in early stages is broadly plexi-
form and has a dorsal and a ventral division.

cor. lym. ant., cor lymphaticum anterius; the surrounding lymphatics are
severing connection with the heart; the lymphaticovenous junction is on the
ventral surface of the heart, and anterior to this are two small venous branches
which later become consolidated with it to form the anterior vertebral vein.

v. jug. ext., vena jugularis externa.

d. Cuv., ductus Cuvieri.

gan. jug. (vag.), ganglion jugulare of the vagus group.

Other structures as in the preceding plates.

126

DEVELOPMENT OF LYMPHATICS IN

OTTO Fo KAMPMETIER

PLAT 6

temp. Si.
max.prim.

lym.ju

PLATE 7
EXPLANATION OF FIGURE

34 Reconstruction of the vascular channels and other structures in the
region of the left pronephros in an 8-mm. embryo of Bufo vulgaris (K. E. C.,
series B 49), lateral view; X 166.

lym. jug., lymphatica jugularis; the point marked * still shows a minute
vestigial connection with the pronephric sinus.

temp. s. max. prim., temporal division of the primary maxillary sinus.

lym. lat., lymphatica lateralis (both dorsal and ventra! divisions) ; the common
channel of the jugular and the lateral lymphatic dorsal to the lymph heart is
referred to in the text and photomicrographs as the common segment of these
vessels.

cor. lym. ant., cor lymphaticum anterius; the surrounding lymphatic plexus
has temporarily severed ts connection with the heart.

v. vert. ant., vena vertebralis anterior.

v. jug. int., vena jugularis interna.

v. jug. ext., vena jugularis externa.

v. card. post., vena cardinalis posterior.

d. Cuv., duetus Cuvieri.

med. spin., medulla spinalis.

gan. jug. (vag.), ganglion jugulare of the vagus group; vag. (ef. figs. 31-83).

Other structures as before.

128

DEVELOPMENT OF LYMPHATICS IN ANURA
OTTO F. KAMPMEIER

PLATE 7

VAN Lon al Cll

is
oe
Se
fol
a
rae)
&

.

v.catd.posi.
d.proneph.

cor lym.ant.

129

PLATE 8
EXPLANATION OF FIGURE

35 Reconstruction of the vascular channels and other structures in the
region of the left pronephros in a 10-mm. embryo of Bufo vulgaris (K. E. C.,
series B 34), lateral view. X 166.

cor. lym. ant., cor. lymphaticum anterius; the lymphaticovenous junction or
efferent portal is shown at e/.; af., indicates the point at which the afferent portal
is being established.

lym. jug., lymphatica jugularis.

lym. lat., lymphatica lateralis.

temp. 8. max. prim., posterior portion of the temporal division of the primary
maxillary sinus.

v. vert. ant., vena vertebralis anterior.

v. jug. int., vena jugularis interna.

v. card. post., vena cardinalis posterior showing both the medial (subeardinal)
and lateral divisions.

d. Cuv., duetus Cuvieri.

d. proneph., pronephric duct.

Other structures as before.

130

YT OF LYMPHATICS IN ANURA

PLATE 8
OTTO F. KAMPMEIER

med. spin.

v.vertant,

in

V JUS,

x
cs
5
2
ioe
=
0)

+

corlym. | lym.lat.
ant.

V.card. post.

tesumen por el autor, Halsey J. Bagg

Trastornos en el desarrollo de los mamiferos producidos por
las emanaciones de radio.

In un grupo de ratas adultas el autor las sometié antes y
después de aparearse a las inyecciones de pequefias dosis de solu-
ciones radioactivas. Las inyecciones fueron subcutdneas e
intravenosas. En-un segundo groupo ratas prefiadas, casi a
término, fueron expuestas a la radiacién de rayos gamma fuerte-
mente filtrados. La dosis ordinaria fué unos 1350 milicuries
horas. Generalmente una cantidad de emanaciones de radio
inicialmente equivalente a mas de un gramo de bromuro de
radio fué empleada durante pr6ximamente una hora.- Los ecam-
bios caracteristicos producidos por el radio fueron hallados en
las erfas de las hembras previamente inyectadas con soluciones
radioactivas. Estos cambios fueron dreas subcutdneas de
extravasacion, situadas principalmente a lo largo de la linea media
dorsal de los embriones. Es interesante notar que en tres crias
diferentes se encontraron lesiones muy separadas en los jOvenes
de madres que fueron tratadas varios dias antes de la fecunda-
cion. Muchos de estos cambios pueden notarse en embriones
expuestos a la radio-actividad después de haber comenzado el
desarrollo, pero generalmente los embriones murieron durante
el tratamiento y fueron mds tarde reabsorbidos o abortados.
Las ratas JOvenes tratadas con la radiacién de los rayos gamma
tres dias antes del nacimiento presentaban el mismo tipo de
lesiones indicadas mas arriba, pero en adicién se encontraron
mareadas interrupciones en el caso del sistema nervioso central,
el sistema reproductor, y especialmente en la formaci6n de los
ojos. Varios de estos animales carecian practicamente de neo-
palio, pero salvo por el hecho de ser ciegos no presentaron rac-
ciones neurol6gicas anormales. En este grupo varios animales
vivieron durante mas de un ano y erecieron hasta aleanzar el
tamanho normal.

Translation by José I. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 27

DISTURBANCES IN MAMMALIAN DEVELOPMENT
PRODUCED BY RADIUM EMANATION

HALSEY J. BAGG

Huntington Fund for Cancer Research, Memorial Hospital and the Department
of Anatomy, Cornell University Medical College, New York City

TEN TEXT FIGURES AND THREE PLATES (FIGURES ELEVEN TO FIFTEEN)

The effects of radium on animal development has been the
subject of several researches since the early work of Bohn (1),
in 1903, upon the ova and larvae of the sea-urchin. ‘ Experiments
on developing nematodes, molluscs, amphibians, fishes, and birds
are associated with the names of Perthes (2), P. Hertwig (3),
Schaper (4), O. Hertwig (5), and G. Hertwig (6). These investi-
gators report developmental retardations following radiation of
the ova and developing embryos. They found a particular sus-
ceptibility of the nuclei of the cells and a general slowing up in the
developmental processes, especially in the case of the central
nervous system. The total disturbances, depending upon the
period of development when the radiation was applied, resulted
in the formation of monstrosities conforming more or less to a
general type.!

Similar experiments concerning the effects of x-rays on develop-
ment have been conducted by many investigators. After ex-
posure to x-radiation, Perthes (7) noted abnormal cell division
and a retardation in the development of the ova of Ascaris mega-

1 In connection with the above statement, and applying to x-ray treatments 4s
well, the question of dosage is an important one. A survey of the literature shows
that there was a very wide range in the severity of the dose employed, and in sev-
eral cases the experimental settings were inadequately described (Bohn used
‘some centigrams’ of pure radium bromide for from twenty minutes to two hours).
The amount of radium metal used in the investigations that have been mentioned
varied from 2 mg. to 35.1 mg., and the time from a few seconds to several hours.
The deleterious changes in the animal tissues varied with the amount of radium
and the time of exposure.

133

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, No. 1

134 HALSEY J. BAGG

locephala. Gilman and Baetjer (8), after radiating the ova of
Amblystoma, and Baldwin (9), the fertilized ova of frogs, were
able to produce a fairly constant type of developmental defect.
Injurious results have followed in all cases where mammals have
been exposed to x-radiation. It has been shown that when any
particular part of a young animal is exposed to a sufficient amount
of radiation, that part fails to reach its normal size and is unable
to exercise a full degree of function.

Arrests in development and the production of abnormal types
may be induced not only by radio-activity, but by many physical
or chemical agents. Abnormal temperature changes, treatment
by many chemicals, lack of oxygen supply, or the overabundance
of carbon dioxide, ete., have produced marked changes in the
developing embryo.

The present experiments are mainly concerned with distur-
bances in mammalian development, before and after birth, as a
result of exposing the embryos of rats, at various times during
the prenatal period, to irradiation from radium emanation. The
effect on the embryos following radiation of the mother at varying
intervals before mating was also determined. These experiments
were designed not only to study the factors underlying the pro-
duction of abnormal types, but through an examination of the
abnormal to gain a clearer insight into the nature of normal
development and differentiation.”

I acknowledge with pleasure my indebtedness to Dr. James
Ewing for his aid in the interpretation of the pathological results.

METHODS AND APPARATUS

Two methods were used for applying the radium emanation.
In the first method an ‘active deposit’ was obtained by exposing
a definite quantity of common salt to a comparatively large
amount of radium emanation, about 500 millicuries were used,

? Dr. J. F. Gudernatsch was a co-investigator with the writer during the year
1919. A preliminary report of the work done with him at that time is given in
the Proceedings of the Society for Experimental Biology and Medicine, 1920,
vol. 17, p. 183.

OO

RADIUM IRRADIATION AND DEVELOPMENT 135

or the amount of radium emanation initially equivalent to one-
half a gram of radium metal. To the radio-active salt thus pro-
duced sufficient water was added to make a physiological solu-
tion. The pregnant rats were injected subcutaneously in the
shoulder region and intravenously through the caudal vein; 3 to 4
minims constituted the usual dose. Because of the rapid loss of
radio-activity of these solutions, the injections were made im-
mediately after the preparation. The details involved in pre-
paring and measuring the doses, as well as the methods for pro-
tecting the experimenter, are described elsewhere (16 and 11).
The activated solution exhibited all the known phenomena of
radium metal itself; alpha, beta, and gamma rays were present,
but the greatest physiological effects were probably due to alpha-
ray activity. After long experimentation, a dose of 5 millicuries
was found to be the maximum applicable to the aims of this experi-
ment. In the second method gamma-ray radiation was applied
through the ventral body wall of pregnant rats at nearly full term.
A large amount of radium emanation was used, an amount equiv-
alent to 14 grams of radium metal, filtered by 2 mm. of lead and
3 mm. of silver. The source of emanation was 1 cm. away from
the animal. The applicator, called a ‘lead tray’ in clinical usage,
was 6 cm. in diameter and 1.5 em. high. This was placed in the
bottom of a small wire cage, 10 by 13 em. in diameter and 10 cm.
high, and was covered by a thin sheet of cardboard. The animal
was placed on this paper immediately above the applicator.

Preliminary tests showed that a dose of about 1300 millicurie
hours was sufficient to produce developmental arrests in the
embryos without killing the pregnant animals. Doses as high as
2900 me. hrs., however, were successfully used in some cases.
The embryos were killed by ether, and histological material pro-
cured at various periods after the treatment. The tissues
were fixed in Bouin’s solution, cut in serial section, and stained
with haematoxylin and eosin.

136 HALSEY J. BAGG

EXPERIMENTAL RESULTS
Series A. Injections of radio-active solutions

I. Subcutaneous injections after mating. Sixty-five full-grown,
normal, pregnant rats were treated in this series. They were
divided into four groups, each treated at different periods after
mating. ‘Ten pregnant females were injected 7 days after mating;
twenty-four, 10 to 14 days after; twenty-one, 15 to 17 days after;
and ten, 18 to 21 days after mating. Many of the animals were
killed at weekly intervals after treatment, although some were
allowed to reach full term.

Various degrees of developmental disturbances were noted, as
shown in the following groups:

1. There was a large number of cases where no embryos
developed, in others many began development, but were absorbed
or aborted at an early time. The females in which no embryos
were found, although they were definitely considered pregnant
before treatment, occurred among cases treated soon after mating
and in those instances where females were autopsied a consider-
able time after treatment. Figure 1 shows the remnants of
maternal and embryonic structures; from the size of the placentae
one can see that the foetuses had reached a fair degree of develop-
ment before the radiation retarded the normal physiological
processes. In one case (fig. 2) a small ovoid sac was found
attached to the uterine wall by a thin stalk. This apparently
represented the remnants of a former embryo and placenta.
Extravasated blood and cell detritus were found in this sae and
a great many large cells of an epithelioid nature that probably
belonged to the former embryonic syncytium. The wall of this
cyst was formed by fibrous connective tissue.

2. Embryos were killed by the treatment, but were removed
from the mother and preserved before they were absorbed.
These showed various extravasations from the vessels of the
subcutaneous connective tissue, within the meningeal sinuses, and
mainly along the dorsal midline of the body. Figure 3 shows a
typical example of such a lesion which was situated in the mid-
dorsal line. The mother of this embryo, no. 1167, was mated on

RADIUM IRRADIATION AND DEVELOPMENT Lar

April 22, 1919, injected with 4.9 millicuries on May 7th, and was
killed two days later. When the embryo was cut in serial section,
it showed that the haematoma in the dorsal subcutaneous tissues
had exerted sufficient pressure upon the spinal column to produce
at one place a complete dislocation. Microscopical examination
of the viscera showed no pathological changes. Not all the
foetuses of a litter were affected in the same degree. In one case
seven foetuses were found, three showing haemorrhagic lesions,
two beginning to macerate, and two in the process of absorption.
This variation in resistance was due either to the higher or lower
vitality of the embryos themselves or to the amount of radio-
activity which passed the placentae. In another case the foe-
tuses, although injured, were carried to full term, and among a
litter of six young, two were apparently normal and four showed
haemorrhagic spots on the head, face, and along the dorsal mid-
line of the body.

3. Several young of a single litter showed areas of extravasa-
tion and were born alive. Their mother died, however, and
foster mothers refused to nurse them.

4, Eight litters gave normal living young. This number is
low, because, as previously stated, many pregnant rats were
killed by the experimenter at various intervals after treatment.
The average number of young per litter was 4.8, as compared
with 6.5 per litter for the control rats, but the probable errors
indicate that this difference is, very likely, not significant. Only
one litter, containing four young, survived a treatment given
seven days after mating. Several of the rats of this group,
which had apparently escaped the full radium exposure during
the uterine period or perhaps they were more resistant to it, when
mated inter se produced litters of apparently normal young of
normal fertility. The offspring of these animals, about twenty
in number, were observed for two generations, but no abnormal-

' ities were noted.

II. Subcutaneous injections before mating. Seventy-seven fe-
males were treated in this group, eleven died as a result of the
injection before they were mated, while several were killed at
weekly intervals after mating, and some were allowed to continue

138 HALSEY J. BAGG

Fig. 1 Two well-developed placentae are shown at the left attached to a uterus
which has been partly opened. The remnants of embryonic tissue are superim-
posed on the placentae. At the right is a placenta which has been dissected from
the uterus, and shows more clearly the remains of embryonic material, here

RADIUM IRRADIATION AND DEVELOPMENT 139

to fullterm. Thirty-four animals were injected between 5 and 7
days before mating; seventeen, 10 to 14 days before, and fifteen,
20 days before mating.

Only three litters in this group showed abnormal young. The
most interesting was a litter of seven, in which case the female
was treated with 4.2 mc., 22 days previous to fertilization, and
the foetuses, approximately 16 days old, showed very pronounced
areas of extravasation, which in one case (fig. 4) covered a large
area on one side of the head and a few small scattered areas on
the other side. ‘These areas were not only along the dorsal mid-
line, but also on the lateral surfaces of the body as well (fig. 5).
The lesions were much more widely distributed and more variable
in size than in the cases recorded under section I. Although the
conditions that produced these results were repeated many times,
the above is the only case where positive data were obtained.
Usually the female had either been rendered sterile or the young
were killed and absorbed during early stages. There were two
other cases, however, where young were found with haemorrhagie
areas, and these occurred in a group of females that were treated
seven days before mating. Female 85 was given a dose of 6.6 me.
on November 7, 1919. It was mated on November 14th, and

represented as a lighter area in the upper portion of the drawing. Female mated
April 22, 1919, injected May 7th, killed May 16th. Dose = 4.6 mc. (subeutane-
ous).

Fig. 2 A stalked sac partly dissected from the uterus, showing the remnants
of a former embryo and placenta. Female mated April 22nd, injected May 7th,
killed May 16th. Dose = 4.8 mc. (subcutaneous).

Fig. 3. This is a dorsal view of a rat embryo, showing a characteristic area of
extravasation due to the treatment of the mother during pregnancy. Female
mated April 22nd, injected May 7th, killed May 9th, at which time seven foetuses
were found about fifteen days in development. Two of the litter were macerated
and two absorbed. Dose = 4.9 me. (subcutaneous).

Fig. 4 Areas of extravasation are shown in the two views of this embryo,
similar to the condition shown in figure 3, but in this case resulting from treating
the mother twenty-two days before fertilization. There are a few small scattered
areas over the right side of the head and a large area of extravasation on the left
side. Female injected April 22nd, mated May 12th, killed May 30th. Seven
foetuses were found, fifteen to sixteen days old. Dose = 4.2 mc. (subcutaneous).

Fig. 5 These are three views of another foetus, a litter mate of the one shown
in figure 4, showing the wide distribution of the extravasated areas over both sides
and back of the animal. The experimental conditions are the same as for figure 4.

140 ' HALSEY J. BAGG

as three young were born December 11th, fertilization took place
about fourteen days after the treatment. ‘Two of the young were
apparently normal, but one showed a large haemorrhagic area,
which involved most of the right side of the snout, the right eye,
and a portion of the lower jaw on that side. This area disap-
peared after three days. Female 99, injected and mated at the
same time with female no. 85, received a dose of 5.6 me. Five
young, three males and two females, were born on December 13th,
making the date of fertilization about sixteen days after treat-
ment. One male and one female showed definite haemorrhagic
areas on the face. Consideration of these cases will be deferred
until later.

Seventeen females following treatment were killed at varying
intervals after mating and showed markedly haemorrhagic or
cystic ovaries and congested uteri. In these cases radium emana-
tion apparently had either so altered the maternal tissues as to
prevent fertilization or development when started was soon
followed by the death of the embryo and its absorption. Many
nodules were found in the uteri in which it was impossible to
differentiate between embryonic and maternal structures.

The remaining females (as previously stated, eleven died
between the period of treatment and mating) produced either
full-term normal young or young apparently normal at autopsy.
Several of these living young grew normally and were mated
inter se, but produced no abnormal offspring, although observed
for two generations.

III. Intravenous injections after mating. The intravenous
injections were primarily planned to act as a check on the series
of subcutaneous treatments. The object was to determine the
immediate reactions that might occur in the embryo as a result
of injecting a comparatively large dose of radio-active solution into
the circulation of the pregnant female, and whether these re-
actions would be similar to those already recorded for the sub-
cutaneous series. ‘The toxic reactions were so prompt and fatal
that it was not necessary to treat many animals to settle this
point. A typical case in that of female no. 123. ‘I his animal,
of about nineteen days’ pregnancy, was treated with 30 me.

RADIUM IRRADIATION AND DEVELOPMENT 141

injected directly into the blood stream through the caudal vein.
This was six times greater than the usual dose in the first twoseries.
Three young were born dead twenty-four hours later. They
showed very definite radium changes, typical of those already
recorded for the subcutaneous series. Figure 6 shows a foetus
still attached to an apparently normal placenta, but a characteris-
tic area of extravasation was found over a considerable portion
of the left side of the head. In figure 7A a dorsal view shows
another embryo with two comparatively small haemorrhagic
areas along the dorsal midline, and the placenta in this case is
also normal. The third foetus in this litter was apparently
normal, but the placenta (fig. 7B) had acted in the nature of a
‘shock absorber’ in protecting the foetus from exposure to the
radio-activity, and it was so swollen and completely filled with
blood as a result of its injury, that it had the appearance of a
large haemorrhagic sac.

Series B. Results from radiating nearly full-term pregnant rats with
gamma-ray radiation

Ten rats were treated at the end of about nineteen days of
pregnancy. It was found that exposure to about 1350 mc.hrs.
of radium emanation was sufficient to produce very decided
changes in the embryo and yet leave the pregnant females suf-
ficiently uninjured to be able to nurse their young and care for
them until after the weaning period. When the dose was
increased to 3378 me.hrs., the young were severely injured, and
were either killed outright or died two or three days after birth.

The following are the conditions that resulted in the first
generation of animals treated in utero with a dose of about
1350 mce.hrs.:

1. The young of each litter were born two or three days after
the treatment, alive and apparently normal.

2. About ten days after treatment, about half of each litter
became markedly anemic, showed symptoms of diffuse edema,
and promptly died. There was an easily recognizable slow
development of meningeal and spinal-cord haemorrhages, similar
to those already described as a result of treatment by radio-active

Fig. 6 There is a large and a small area of extravasation on the head of this
foetus. The mother was injected intravenously with 30 me. of radio-active
solution and three young, about full term, were born twenty-four hours later.
All were dead. In this case the attached placenta is apparently normal.

Fig. 7 The lower figure (A) shows an embryo with two dorsal head lesions
and an apparently normal placenta. This is a litter mate of the animal shown in
figure 6. At B is indicated a large haemorrhagic placenta from the third young
of this litter, which itself was apparently normal.

Fig. 8 Dorsal view of a young rat with the skin dissected to either side.
There is a prominent area of meningeal extravasation in the frontal region.
This animal was treated in utero with 1350 mc.hrs. of gamma-ray radiation on
TFebruary 21st and was born apparently normal on February 23rd. It died on
March 3rd.

Fig. 9 Dorsal view of a young rat showing areas of frontal and occipital extrav-
asations which were within the meningeal sinuses. There is a smaller lesion
in the left dorsal thoracie region. This is a litter mate of the animal shown in
figure 8, and the experimental conditions were identical. Death occurred on
March 3rd.

Fig. 10 There is an extensive meningeal extravasation over a considerable
portion of the hemispheres, and a haemorrhagic lesion is shown on the reflected
skin from the dorsal interscapular region. This is a litter mate of the aniraals
shown in the two preceding figures. Death occurred on March 2nd.

142

RADIUM IRRADIATION AND DEVELOPMENT 143

solutions. A series of these lesions is shown in figures 8, 9, and 10.
Figure 8 shows a young rat with the dorsal integument partly
dissected away, exposing a typical haemorrhagic area in the region
of the frontal lobes. The slow development of this lesion could
be easily noted through the thin, transparent scalp. This young
was one of several treated in utero with 1350 mc.hrs. of gamma-
ray radiation on February 21, 1920. It was born two days later,
and died on March 38rd. The young rat shown in figure 9 was
a litter mate of the previous animal. It shows the presence of
three distinct haemorrhagic areas, a small frontal lesion, a fairly
extensive one in the occipital region, and a small lesion in the
subcutaneous tissues in the thoracic region, near the middorsal
line on the left side of the body. This animal also died on
March 8rd. _ A third animal belonging to the same litter is shown
in figure 10. Here is seen a still more acute reaction, as shown
by the fact that the animal died a day sooner than in the two
cases above. There is an extensive area of meningeal haemor-
rhage which covers most of the dorsal portion of the brain,
involving the frontal and occipital regions and the medial area
between, as well as a considerable portion of the right temporal
area. In addition, a distinct, rounded haemorrhagic lesion may
be noted on the reflected skin on the left side of the body. This
lesion occurred in the midshoulder region of the back.

The heads of several of the young rats showed marked lateral
compression. In one case a haemorrhage so affected the spinal
cord as to produce complete paraplegia. The tissues of these
animals were studied histologically. Save for the mechanical
disturbances produced by the presence of the extravasated areas,
the most marked pathological conditions were seen in the liver
and intestines. In the first case there was a pronounced fatty
degeneration of the hepatic cells, and in the second, a desquama-
tion of the lining cells of the intestinal mucosa.

3. It is interesting to note that the other half of each litter
survived the treatment, grew to a normal size, and some ani-
mals have lived for over eighteen months. They showed the
effects of the late uterine treatment by the following arrests in
development: ,

144 HALSEY J. BAGG

a. The first pathological condition noted was that the eyes
became smaller, the pupils opaque, and there finally was a com-
plete, or nearly complete, closing of the lids and total blindness.
This condition was first observed a short time after the eyes had
opened. The photographs in figure 11 show three views of a
female rat about one year old with typical eye deformities. ‘The
upper view shows the entire animal, which had grown to normal
size and weight for its age. The left eye was nearly completely
closed, as is shown more clearly in the lower right-hand view of
the head at a higher magnification. Both pupils were opaque,
but, as shown in the illustration, the right eyelids were slightly
more opened than those of the other side. ‘The animal was one
of a litter treated in utero on March 8, 1920, was born six days
later, and the photograph was taken on March 1, 1921. The dose
in this case was 2920 mc.hrs. of gamma-ray radiation, which was
a dose higher than that usually tolerated.

b. Mating tests showed that both the males and females were
completely sterile in the first lots, but subsequently a first-genera-
tion female, that had been treated with 1350 mc.hrs., mated
with a male similarly treated, gave birth to nine apparently
normal young.

c. Before these adult offspring of treated animals were killed
for histological examination, their neurological reactions were
very carefully studied. The animals, being blind, when startled
assumed various defensive attitudes, but save for these reactions
their behavior was remarkably normal. There was no ataxia
in locomotion or in any of the feeding reactions, auditory acuity
was normal, and there was no cutaneous hypoesthesia or other
sensory disturbances. Except for blindness, there was nothing
to suggest abnormal sensory function.

d. When these animals were autopsied, marked developmental
disturbances were noted in the condition of the central nervous
system. The cerebral hemispheres were greatly reduced in size,
and in several cases very little cortical material remained. ‘Those
portions of the brain that were ontogenetically older (the archio-
striatum and the cerebellum) were apparently normal. The
optic tracts were markedly atrophic. Correlated with this

RADIUM IRRADIATION AND DEVELOPMENT 145

disturbance in brain development, the skull was found to be
asymmetrical, narrow, thicker than normal, and concave in the
frontal region.

Figure 12 shows a dorsal view of a normal, untreated brain of
an adult rat, magnified five diameters. In figures 13 and 14 are
dorsal and lateral views of a brain of oneoftherats which belonged
to the same litter as those of section 2 of this series. ‘This animal
was treated with 1350 me.hrs. on February 21, 1920, was born on
February 23rd, and was killed December 31, 1920. This was one
of the animals which (except for blindness) showed no abnormal
neurological reactions. The magnification in figures 13 and 14
is the same as that for the control brain in figure 12. The dorsal
view in figure 13 shows an apparently normal cerebellum and
normal olfactory lobes, but the part of the brain which represents
the rudiments of the hemispheresshowsa great lack of development
of cortical substance. In a side view of the brain in figure 14,
the cortex may be seen to be very thin; indeed, not completely
covering what should normally be the frontal, occipital, and
lateral aspects of the brain. The remains of the hemispheres do
not sufficiently approach each other in the median line to cover
the colliculi beneath. In figure 13 the meninges on the left side
of the brain have been removed, but on the other side they have
been left in place. It was possible in this specimen to see the
lateral ventricles through the transparent membranes. Several
other brains have been studied which showed various degrees
of developmental arrests resulting from radium treatment. In
some cases the hemispheres were markedly reduced in size, were
widely divergent in the median line, and yet the pallium was
complete over the entire surface. In all these cases there was
marked optic atrophy. These brains are now being sectioned,
and a study of them in greater detail will be the subject of a
separate communication.

e. A histological study of the eye showed that the eyeball was
reduced to one-fourth the normal diameter. The retina was
missing, but traces of the choroid remained as a few scattered
pigment cells. The cornea was three times as thick as normal
and covered with four or five layers of opaque squamous epithe-

146 HALSEY J. BAGG

lium. The optic nerve was extremely small, not more than one-
seri? the normal dimensions.

. The testes of the radiated animals were decidedly atrophic,
zs a comparison with the normal is shown in the photograph
in figure 15. The diameters of the testicle alone (minus the
epididymis) of the experimental animal was 14 mm. for the length
and 7 mm. for the width, while the control measurements from
normal animals of the same age and weight and with the same
method of fixation were 21 mm. for the length and 11.5 mm. for
the width. ‘The epididymis of the radiated testis was practically
missing. A small portion of the tail remained, but the head and
body of the epididymis had failed to develop. Histological
examination shows that there is little evidence of spermatogenesis.
Some tubules seem to contain imperfect spermatoblasts and
forming spermatozoa, but the great majority of tubules show
complete degeneration and loss of epithelial cells, and contain
loose granular material, which in places is calcified. Some
spermatic tubules are greatly dilated and filled with granular
material. Very few interstitial cells are visible.

The ovary of the radiated animals was reduced to one-fourth
or one-fifth the normal size. The graffian follicles were entirely
missing. “Groups of lutein cells persisted in small numbers, but
showed marked hydropic degeneration. Some of the large
vessels about the ovary were sclerosed.

g. The liver, kidney, lungs, spleen, and the other organs were
examined, but showed no pathological disturbance.

CONTROL GROUP

Pregnant rats of the same stock, the same age and weight,
were injected subcutaneously and intravenously with equal
amounts of solutions that previously had been strongly radio-
active, but were allowed to ‘decay,’ until they had lost their
radio-activity. These experiments gave absolutely negative
results. As a control to the gamma-ray experiments, pregnant
rats, sisters of the treated animals, were allowed to breed under
exactly the same experimental conditions. No abnormal young
were observed.

RADIUM IRRADIATION AND DEVELOPMENT 147
DISCUSSION AND SUMMARY OF RESULTS

It has been shown that when doses of radio-active solutions
are injected into an animal marked physiological reactions take
place. Large doses produce severe toxemia, resulting in pro-
nounced pathological changes in the various viscera of the white
rat (10). A study of metabolic changes in dogs, as determined
by urine analysis, showed that, following intravenous injections
of such solutions, there were very decided increases in the total
nitrogen content of the urine, the urea, creatinine, uric acid, and
the total phosphates (12). A prompt reduction occurred in the
number of white blood cells of the dog after intravenous injections
of these solutions, associated with a marked decrease in the
relative percentage of circulating lymphocytes (13). In order
to reduce as much as possible the severity of the reaction, very
small doses of radio-activity were used in the experiments
recorded in this article. But even with comparatively small
doses, certain rats treated in utero showed very acute reactions.
Many were killed by the treatment and were absorbed or aborted.
Others were found showing pronounced areas of subcutaneous
extravasations, mainly situated along the middorsal line of the
body and within the meningeal sinuses. This condition was
probably due to the destructive action of radium on the endothe-
lium of the blood vessels, as well as a possible increase in blood
pressure, as was shown to occur in the dog by Burton-Opitz and
Meyer (14) after intravenous injections of very small quantities
of radium bromide. A similar reaction of the blood vessels to
radiation was previously reported by Halkin (15) for the skin of
pigs, and by Danysz (16) for radiated mice. This destructive
action of radium on the blood vessels is in line with clinical
observations on the usual prompt regression of very vascular
tumors (the angiomata, in particular) after exposure to irradiation.

The changes in the rat embryos of this experiment are inter-
esting in so far as they show that a sufficient amount of radio-
activity was able to pass the placenta and subsequently affect
the developing embryo. This occurred after subcutaneous as
well as intravenous injections of the mother. By far the most

148 HALSEY J. BAGG

interesting observation concerned the presence of lesions similar
to those described above in rat embryos whose mother was
treated with radio-active solutions a considerable time before
mating. The writer has no explanation to account for this
phenomenon. It would appear that the treatment of the mother
several days previous to conception has lessened the faculty of
the later-developing embryo to form proper endothelium of the
blood vessels, and the wide distribution of these lesions over the
body of the embryo (peculiar to this group of animals) would
tend to substantiate this view. One female was injected twenty-
two days before fertilization, and since the solutions lose their
radio-activity very rapidly (there is about a 50 per cent reduction
in the first hour after the preparation) the likelihood of any
radio-activity remaining over during this period and affecting the
egg at a later critical moment is remote. The amount of radio-
activity remaining after twenty-two days, if present at all, should,
as determined from physical computation, beinfinitesimally small.

The series of intravenous injections again emphasize the
specific action of radium emanation in the production of typical
areas of subcutaneous emtravasations in the developing young,
and in addition shows that the placenta may act in the nature of
a ‘shock absorber’ and prevent the embryo from receiving the
full effect of the radiation.

We now come to a consideration of the cases wherein pregnant
females were treated with external applications of comparatively
large doses of gamma-ray radiation. At this time a report is
given only for embryos treated towards the end of pregnancy.
The writer plans to continue this line of investigation and treat
at earlier prenatal periods.

The results emphasize the well-known delayed reaction asso-
ciated with gamma-ray radiation. There was approximately a
ten-day interval following treatment during which no changes
were noted in the embryo, and during this period the young
animals were born in an apparently normal condition. Acute
reactions promptly occurred at the completion of this time,
killing half of each litter. The young rats died showing typical
radium changes, such as anemia, diffuse edema, and meningeal,

RADIUM IRRADIATION AND DEVELOPMENT 149

spinal-cord, and subcutaneous extravasations. These extravasa-
tions were markedly similar to those already described for the
series of solution treatments. The liver in these animals showed
a fatty degeneration of the hepatic cells similar to the condition
reported by Mills (17) after exposing a series of mice to gamma-
ray radiation. The only other pathological change noted in
these embryos was a desquamation of the lining cells of the
intestinal mucosa. This observation is in line with the results
emphasized by Hall and Whipple (18) in their experiments on
Roentgen-ray intoxication in dogs.

While the animals described above died after showing acute
reactions certain of their litter mates (half of the litter) continued
to develop apparently normally. This difference in reaction may
possibly be due to individual variability or tolerance for the
radiation, but it probably can be explained by the fact that cer-
tain embryos were slightly farther away from the source of
radiation than others, and as the intensity of radiation varies
inversely as the square of the distance, even such slight differences
in distances that did exist would be sufficient to subject the
embryos to a considerable range in intensity of radiation. This
is especially important in this case because the source of radiation
was only 1 em. from the body wall of the mother. The quality
of radiation, however, remained the same for all the embryos.

It was soon apparent that the animals that lived over the ten-
day period had not completely escaped the effect of theradiation,
as was shown by a suppression of the full development of the
eyes. Eye defects were noted, such as opaqueness of the pupil,
atrophy of the lens, and closing of the lids, which resulted in
complete blindness. These animals grew to a normal size, suc-
cessfully competed with their cage mates for food, and showed
absolutely no abnormal neurological condition, except those
clearly incident to blindness. At autopsy, in some cases over a
year after birth, very decided developmental arrests were noted
in the structure of the brain. All grades of such maldevelopment
were noted in the condition of the neopallium, from merely a
decrease in the size of the hemispheres, which permitted the
corpora quadrigemina to be clearly visible from above, to a more

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 1

150 HALSEY J. BAGG

marked absence of the cerebral cortex until only a very thin
lamina of tissue remained to represent that structure, and there
were large areas in the frontal, occipital, and temporal region
where no cortex existed at all, so that when the meninges were
removed the basal ganglia were clearly seen from without.

This correlation between defects in the development of the eye
and the brain has been emphasized by Stockard (19) in his recent
paper on developmental rate and structural expression. He
states as follows: ‘‘The periods of arrest necessary to induce
the eye and the brain modifications are so close together or so
nearly the same, that one generally finds combinations and mix-
tures of the defects among the same experimental group of
embryos.’’ Again in the same article Stockard has shown that
the type of deformity that results from experimental disturbance
depends upon the developmental moment at which the imter-
ruption occurs. It is significant that the animals of this experi-
ment showed arrests in the development of the neopallial portions
of the brain and not in those regions which are ontogenetically
older. Apparently the radium emanation, acting towards the
end of pregnancy, had affected the development of the brain
after the basal ganglia, the cerebellum, and medulla had become
fairly well differentiated, and therefore those portions showed no
gross changes. But the radium had slowed the developmental
rate of the neopallium (which we know is one of the last portions
of the brain to differentiate) during its period of active cell pro-
liferation, and that portion of the brain was never able to reestab-
lish its proper rate of development in relation to the other parts
of the brain. If the period of treatment had occurred earlier in
prenatal existence, other portions of the brain would probably
have shown disturbances as well. The writer does not believe
that the deformities in the brains of these animals were due to
the early production of vascular disturbances later recovered
from, but to an actual inhibitory effect of the radiation upon the
developing nerve cells. If extravasations had occurred in this
group of animals, and were so situated as to affect the develop-
ment of the cerebral cortex in particular, they probably would
have been detected as were even the comparatively small lesions |

RADIUM IRRADIATION AND DEVELOPMENT 151

which were associated with the acute reactions. Also, if the
effect was largely due to vascular disturbances, from the nature
of the radiation employed, one would expect more generalized
changes throughout the entire brain. However, on the other
hand, Craigie (20), in his recent paper on the relative vascularity
of various parts of the central nervous system of the albino rat,
suggests that ‘“‘the vascularization of the more recently evolved
centers (of the cortex cerebri) is more susceptible than the more
ancient regions to sexual, hereditary or environmental influences. ”’

From a neurological point of view, it is interesting to consider
that the animals with practically no cerebral cortex reacted so
normally in their ordinary behavior. Except for blindness, there
was no other apparent sensory disturbance, and motor coordina-
tion appeared perfect. The physiological functions localized in
the cerebral cortex of the rat were in these animals apparently
transferred to the basal ganglia and other paleokinetic portions
of the brain, showing the remarkable degree of compensation
possible in the mammalian brain when the disturbing element
acts at an early period in its development. In this connection
it is worth mentioning that the radium emanation did not produce
a sudden traumatic effect, as is normally the case with the experi-
mental production of brain lesions, and, in fact, the radium
changes were probably prolonged over a considerable pericd.
This condition favored the establishment of compensatory
reactions, and exists (as shown by the writer in a recent article
(21)) even in the ease of radium lesions experimentally produced
in adult mammalian brains.

The reproductive system completes its development a consid-
erable time after birth, and so it is not at all surprising that
these structures should have shown a considerable amount of
atrophy due to the developmental arrest during the prenatal
period. The ovaries and testes appeared to suffer with equal
severity.

An interesting correlative relation was shown by the fact that
the other viscera (digestive, excretory, ete.) of the animals that
showed marked developmental arrests of the nervous and repro-
ductive systems were apparently normal and the animals grew

152 HALSEY J. BAGG

to an average size. The lungs, liver, kidneys, ete., had dif-
ferentiated before the physical agent was employed. Further
studies with earlier prenatal treatments should throw some light
on establishing the critical growth periods of the various embry-
onic structures.

As a final point, the results of this investigation may be of
interest to the clinicians and the laboratory workers who handle
large quantities of radium and utilize x-rays. Although the
results of this paper deal only with irradiation of the female, there
is no reason to believe that the germ cells of the male are more
resistant to these destructive agents than those of the female,
and, in fact, there is very good experimental evidence to show that
spermatozoa of some animals are especially likely to produce
abnormal young after exposure to comparatively small quantities
of irradiation. Physicians should guard against the possibility
of producing developmental arrests such as shown in this article
when treating pregnant women, as well as. the possibility
of altering the human germ cells by irradiation previous to
conception.?

CONCLUSIONS

1. The marked selective action of radium emanation on fast-
growing embryonic structures was noted in these experiments.

2. Very decided developmental arrests occurred in the dif-
ferentiation of the nervous and reproductive systems of mamma-
lian embryos exposed to irradiation towards the end of pregnancy.

3. Experimental animals with greatly reduced, or practically
no neopallium, gave apparently normal neurological behavior,
except for blindness.

4, Radium emanation, used either in the form of a radio-active
solution injected into the adult female, or employed as an external

3 The writer does not mean to be understood as stating that present-day clinical
irradiation treatments produce such effects in the developing young, but it is
his personal opinion that such changes are biologically possible. It is not pos-
sible to obtain desired information by comparing the amount of exposure that
a small mammal can stand with the corresponding dose that a man should tolerate,
judging by comparative weights. The small mammal can tolerate very much
more radiation in proportion to its weight than a man can.

RADIUM IRRADIATION AND DEVELOPMENT lao

gamma-ray radiation, produced marked areas of extravasation in
the subcutaneous connective tissue of the developing young.
This suggests that the action of radium emanation might be
selective upon the endothelium of blood vessels.

5. Extravasations occurred in the developing young of females
treated with radio-active solutions a considerable time before fer-
tilization, and suggest that in some way the faculty of the later
developing embryos to form proper blood vascular endothelium
had been interfered with.

6. The results so far obtained indicate that gamma-ray radia-
tion is a physical agent admirably adapted to the study of experi-
mentally produced developmental arrests in mammalian embryos.

7. When women are subjected to therapeutic irradiation,
especially during the early stages of pregnancy, the clinician
should be forewarned concerning the possibility of producing very
grave disturbances in the developing child.

LITERATURE CITED

1 Boun, B. 1903 Action des rayons du radium sur les téguments. Compt.
rend, Soe. de biol., T. 40, p. 1442.

2 PrertHEs, G. 1904 Versuche iitber den Einfluss der Réntgenstrahlen und
Radiumstrahlen auf die Zellteilung. Deut. med. Woch., Bd. 30, 8S. 632.

3 Hertwic, P. 1911 Dureh Radiumbestrahlung hervorgerufene Verinder-
ungen in der Kernteilungsfiguren der Kier von Ascaris megalocephala.
Archiv. f. mikros. Anat., Bd. 77, No. 2, S. 301.

4 Scuapger, A. 1904 Experimentelle Untersuchungen iiber die Wirkung des
Radiums auf embryonale und regenerative Entwicklungsvorgiinge.
Deut. med. Woch., Bd. 30, 8. 1434.

Hertwia, O. 1911 Die Radiumkrankheit tierischer Keimzellen. Archiv.
f. mikros. Anat., Bd. 77, No. 2, S. 1.

6 Hertwia, G. 1911 Radiumbestrahlung unbefruchteter Froscheier und ihre
Entwicklung nach Befruchtung mit normalem Samen. Archiv. f.
mikros. Anat., Bd. 77, No. 2, S. 165.

Prertues, G. 1904 Versuche iiber den Einfluss der Réntgenstrahlen und
Radiumstrahlen auf die Zellteilung. Deut. med. Woch., Bd. 30, 8. 668.

S Gruman, P. K., anp BAartser, F. H. 1904 Some effects of the Réntgen rays

on the development of embryos. Amer. Jour. Physiol., vol. 10, p. 222.
9 Baupwin, W. M. 1919 The artificial production of monsters conforming
to a definite type by means of x-rays. Anat. Rec., vol. 17, no. 3, p. 135.

10 Baae, H. J. 1920 Pathological changes accompanying injections of an
active deposit of radium emanation. Jour. Cancer Res., vol. 5, no.
il, Joy The

or

|

154. HALSEY J. BAGG

19

21

Duane, W. 1917 Methods of preparing and using radioactive substances
in the treatment of malignant diseases and of estimating suitable doses.
Boston Med. and Surg. Jour., vol. 177, no. 23, p. 787.

Tuets, R. C., anp Baca, H. J. 1920 The effect of intravenous injections of
active deposit of radium on metabolism in the dog. Jour. Biol. Chem.,
vol. 41, no. 4, p. 525.

Baaa, H. J. 1920 The response of the animal organism to repeated injec-
tions of an active deposit of radium emanation. Jour. Cancer. Res.,
vol. 5, no. 4, p. 301.

Burton-Opitz, R., AnD Mreyrr, G. M. 1906 Effects of intravenous injec-
tions of radium bromide. Jour. Exp. Med., vol. 8, p. 245.

Harkin, H. 1903 Uber den Einfluss der Beequerelstrahlen auf die Haut.
Archiv. f. Dermat. u. Syphilis, Bd. 65, 8. 201.

Danysz, J. 1903 De l’action pathogéne des rayons et des émanations émis
par le radium sur différentes tissus et différentes organismes. Compt.
rend. Soe. de biol., T. 136, p. 461.

Mixtus, G. P. 1910 The effect of radium on healthy tissue cells. Lancet,
vol. 2, p. 462.

Haut, C. C., anp WuippeLe, G. H. 1919 Roentgen-ray intoxication: Dis-
turbances in metabolism produced by deep massive doses of the hard
Roentgen rays. Amer. Jour. of the Med. Sciences, vol. 157, no. 4,
p. 453.

SrockarD, C. R. 1921 Developmental rate and structural expression:
An experimental study of twins, ‘double monsters’ and single deformi-
ties, and the interaction among embryonic organs during their origin
and development. Am. Jour. Anat., vol. 28, no. 2, p. 115.

Craiciz, E. H. 1920 On the relative vascularity of various parts of the
central nervous system of the albino rat. Jour. Comp. Neur., vol.
31, p. 429.

Baaa, H. J. 1921 The effect of radium emanation on the adult mammalian
brain. Amer. Jour. Roent., vol. 8, no. 9, p. 536.

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PLATE 1

EXPLANATION OF FIGURE

11 The upper photograph shows an adult rat with eye deformities due to
gamma-ray irradiation during the prenatal period. The size and weight are
normal for its age. The lower figures show the lateral views of the head at a
higher magnification. Both pupils are opaque and the left eyelids are nearly
completely closed. Mother treated on March 8, 1920, young were born on March
14th, and photograph was taken on March 1, 1921. Dose = 2920 me.hrs.

RADIUM IRRADIATION AND DEVELOPMENT PLATE 1
HALSEY J. BAGG

pean
ou
“I

PLATE 2

EXPLANATION OF FIGURES

12 Dorsal view of a normal untreated brain of an adult rat.

13 Dorsal view of the brain of an adult rat showing the marked develop-
mental arrest in the formation of the neopallium. The meninges on the left
side of the brain are removed. Mother treated with gamma-ray irradiation on
February 21, 1920, young were born on February 28rd. Brain from radiated
young removed on December 31, 1920. Dose = 1350 me.hrs. (For further ref-
erence see text.) The magnification is the same as for the drawing of the normal
brain.

14 A lateral view of the brain shown in figure 13. This shows the thin-
ness of the remains of the cerebral cortex and its total absence in the frontal and
occipital regions. (For further reference see text. )

158

RADIUM IRRADIATION AND DEVELOPMENT
HALSEY J. BAGG

PLATE 2

PLATE 3
EXPLANATION OF FIGURE

15 At the right is shown a normal untreated testicle of a white rat sur-
mounted by a well-developed epididymis, which is partly obscured by fat. At
the left is a radiated testis showing considerable atrophy. The single small lobe
at the very bottom of the photograph represents the remains of the tail of the
epididymis, the head and body of that part being completely missing. The
animal was treated in utero on February 21, 1920, was born February 23rd, and
was killed December 31, 1920. Dose = 1350 me.hrs. of gamma-ray irradiation.

160

RADIUM IRRADIATION AND DEVELOPMENT PLATE 3
HALSEY J. BAGG

161

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THE AMERICAN JOURNAL OF ANATOMY
vou, 30, No. 2, MARCH, 1922

Resumen por el autor, E. A. Boyden.

El desarrollo de la cloaca de las aves, con especial mencién del
origen de la bolsa de Fabricio, la formacién de un seno
urodeal y la presencia constante de una venta cloacal.

El presente trabajo contiene una revisién del desarrollo de la
cloaca de embriones de pollo desde el tercer hasta el décimoquinto
dia de la incubacién, junto con observaciones sobre el desarrollo
de la cloaca en otras tres especies de aves. Elrasgo mas notable es
la presencia consetante de una ventana transitoria, producida por
la desintegracién de un drea definidamente localizada del epitelio y
su destruccién y absorcién ulterior por los fagocitos, a raiz de
cuyas transformaciones el contenido de la cloaca queda én con-
tacto con el mesenquima durante un periodo de casi venticuatro
horas. En opinién del autor esta particularidad susministra el
tinico ejemplo, en la diferenciacién de un organo hueco, de una
desaparicién parcial de la pared epitelial como rasgo normal y
constante del desarrollo.

La segunda parte de este trabajo trata de la formacién de un
seno transitorio situado hacia la region media del eje principal de
la cloaca, el cual se ha interpretado como una repeticién de la
vejiga dorsal de los reptiles. Esta seccién trata también de varias
anomalias interesantes producidas el la desembocadura de los
conductos wolfianos en la cloaca. Un tercer rasgo interesante es
la presencia constante en los embriones de pollo, de un diverticulo
accesorio de la bolsa de Fabricio, el cual se origina probablemente
mediante irregularidades consiguientes a la formaci6n de la ven-.
tana cloacal. Por medio de este diverticulo ha sido posible
definir el esbozo de la bolsa con mds exactitud que habia sido
hecho hasta el presente y, por consiguiente, ofrecer nuevas ideas
en relacién con su origen filogenético.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 27

THE DEVELOPMENT OF. THE CLOACA IN BIRDS,
WITH SPECIAL REFERENCE TO THE ORIGIN OF
THE BURSA OF FABRICIUS, THE FORMATION OF A
URODAEAL SINUS, AND THE REGULAR OCCUR-
RENCE OF A CLOACAL FENESTRA

EDWARD A. BOYDEN
Department of Anatomy, Harvard Medical School

FORTY-ONE FIGURES

The cloaca of the domestic fowl has been an object of interest
to anatomists since early in the seventeenth century. It was
discovered by Hieronymus Fabricius while investigating the
urogenital apparatus of birds in connection with his pioneer
study of the chick embryo. In his posthumous treatise, entitled
“De Formatione Ovi et Pulli,” Patavii, 1621, he describes as
follows a blind sac lying behind the uterus of the fowl, but empty-
ing into the cloaca close to its external orifice:

A third thing to be noted in the anus is a duplex vesicle,!
which in its deepest part rises up to the os pubis, and is seen
clearly and further back as soon as the uterus, already described,
offers itself to view. Since the vesicle is pervious to the extent
that a passage opens below from the anus to.the uterus itself
and from the uterus into the vesicle, as it were superiorly, the
vesicle being closed at the other end, we have come to the belief

1 The meaning of duplex in this passage is doubtful. The organ itself is never
double. But in two species, a nestling raven (Osawa, 711) and the jay (Jolly, 715),
it has been reported as bilobate. Osawa suggests that perhaps Fabricius may
have had such a case before him when writing his description, but apparently
Fabricius dealt only with the fowl, a species in which a bilobed condition has
never been reported. The bursa is, however, two-walled, consisting of a mucous
membrane and a muscular layer; and the recognized use of duplex to mean stout
or thick, as applied to garments, may have been in the mind of Fabricius when he
used this term. Unfortunately, its form is not recognizable in the woodcut in
which he intended to show it.

163

164 EDWARD A. BOYDEN

that this is the place into which the cock injects his semen, and
forces it in so that it is kept there.

This idea of a receptaculum seminis was discussed at length
by his student Harvey, and by de Graaf, the latter publishing
the first picture of the bursa. Both denied the function ascribed
to it by its discoverer on the ground that it was equally well
developed in both sexes. Beginning with the middle of the
nineteenth century, it was subjected to microscopic. examination
and thereafter repeatedly studied, one group of investigators
(Leydig and his successors) holding that it was purely a lym-
phoid organ; another group (Stieda and his school) maintaining,
on embryological grounds, that it was primarily a glandular
organ. Following Kolliker’s description.of the epithelial origin
of the thymus, in 1879, these views were partially reconciled,
but gave rise to a new discussion as to whether the epithelial
primordium is replaced by invading tissue or whether it is itself
transformed into a reticulum containing lymphocytes. Most
authors since Wenckebach (’88) have held that the epithelium
undergoes transformation without invasion. Recently Jolly
(715), in an elaborate summary of five years’ work on the histo-
genesis, haematopoietic activity, and involution of the bursa of
Fabricius, has advanced the theory that ‘‘the bursa represents
an ancestral glandular organ, a cloacal caecum undergoing re-
gression, which has become invaded by lymphocytes like other
retrograding diverticula (the vermiform process of mammals and
the intestinal caeca of birds), but in which, in view of a new func-
tion, a particular adaptation has taken place between the (per-
sisting) epithelial tissue and the (invading) mesodermal, lym-
_ phoid tissue.”? In recognition of this symbiotic relation, Jolly
would define both thymus and bursa as lympho-epithelval organs.
Up to the present time, however, one must acknowledge that all
attempts to analyze the function of the bursa or to find its coun-
terpart in the hind-gut of other vertebrates have met with only
partial success.

Modern investigation of the cloaca may be said to have begun
with the embryological studies of Gasser (’73—’80) and of Wencke-
bach (’88), who reéstablished the view of Bornhaupt (’67) re-

ewan: pe

THE CLOACA IN BIRDS 165

garding the entodermal origin of the bursa. Since then only one
paper has added any substantial increment to our knowledge of
the general development of the avian cloaca, that of Pomayer
(02), in the Fleischmann series, dealing especially with the
development of the phallus.

The present study originated with the discovery of a temporary
foramen in the dorsal wall of the cloaca, produced by the disin-
tegration of a definitely localized patch of epithelium and its
subsequent removal by phagocytes, following which the con-
tents of the cloaca are left in contact with the mesenchyma for
a period of nearly twenty-four hours of incubation. This curious
phenomenon was observed in over thirty embryos of the Harvard
Collection, and its failure to occur has not been recorded in any
embryos incubated approximately three days, the period at which
the fenestra reaches its maximum size. My attention was
first attracted to it by the presence of large numbers of embryonic
phagocytes? similar to those found in the vestigial gill-filaments
of chick embryos of corresponding age (Boyden, 718). Further
study then demonstrated that this peculiar foramen was con-
stant in its mode of development and invariably occurs, not only
in chick embryos where it was first found, but in duck and pheas-
ant embryos as well. It is of special interest not merely because
it furnishes the only instance in the differentiation of a hollow
organ, so far as I am aware, in which a gap occurs in the epithe-
lial wall as a normal and constant feature of development, but
also because it enables us, by virtue of the landmarks it estab-
lishes, to determine for the first time the exact point of origin of
the bursa of Fabricius.

2 These cells were first described as degeneration cysts, but they were subse-
quently seen in the underlying tissues into which they had been extruded from the
epithelium, and were then recognized as embryonic phagocytes. It isa debated
question whether these should be classed with the wandering cells of later embry-
onic stages and thus derived from the mesenchyma in general (the macrophages,
clasmatocytes, etc., of numerous authors), or should be considered to have arisen
in situ as reactions of the local mesenchyma, or even of the epithelium itself, to
the presence of dead protein. This problem will be discussed in another paper in
connection with the appearance of phagocytes in the anal plate at so early a period
as forty-eight hours of incubation.

166 EDWARD A. BOYDEN

In following the origin and fate of this particular foramen, to
which I have applied the name cloacal fenestra, it became neces-
sary to review the entire chain of events in thedevelopment of the
cloaca from the formation of the primitive streak to the period of
histological differentiation, and to supplement a quantitative
study of chick embryos with observations on other species,
notably duck, pheasant, gull, and tern embryos. As a result of
this study a number of other interesting facts have come to
light. Those relating to the early development of the hind-gut
and tail have been reserved for a subsequent publication.

DEVELOPMENT OF THE CLOACAL FENESTRA >

In describing the origin of this foramen it will be necessary to
refer occasionally to a peculiar tissue in the sacrocaudal region of
young chick, pheasant, and duck embryos, which up to this time
has not been observed in other birds or vertebrates. I refer to
an indifferent cell-mass in the proximal end of the tail which
persists long after the adjacent region has been differentiated—as
late as the beginning of the fourth day of incubation in chick
embryos. As seen in figure 5 (ps. v.), this inert mass lies within
the angle formed by the cloaca and the caudal intestine, to both
of which structures it is fused in a sagittal plane. Laterally it
passes over into the mesenchyma of the tail, but rather abruptly,
so that its limits can be approximately defined and the whole
mass modeled in relation to surrounding structures, as displayed
in figure 13. Beginning at the proximal end of the tail, this tis-
sue is seen to be directly fused with the wall of the cloaca in the
territory included between the anal plate and the junction of the
caudal intestine with the cloaca (this being the wall of the cloaca
which will later give rise to the bursa of Fabricius). Dorsally,
this tissue is fused with the ventral border of the caudal intestine,
and so intimately that the latter never has a chance to differen-
tiate into an epithelium before it is resorbed. Ventrally, it
fuses with the ectoderm bordering the anal sinus, while caudally
it merges with the tail-bud mass—a fusion of three germ-layers
extending across the tip of the tail. Thus the core of the tail
is composed of an indifferent cell-mass, the whole of which can

THE CLOACA IN BIRDS 167

GRAPHIC RECONSTRUCTIONS ILLUSTRATING INITIAL STAGES IN THE FORMATION
OF THE CLOACAL FENESTRA

(Dotted lines and arabic numerals refer to somites; dash lines, to cavities of
the cloaca; crosses, to the primitive-streak mass; periods, toscattered phagocytes;
cross-hatching, to concentrated areas of disintegration on left side of embryo.)

Fig.1 Tern embryo (Sterna hirundo) H.E.C. 2167:5. mm. X 42. all.,
allantois; an. pl., cloacal membrane; c. 7., caudal intestine; rect., rectum;-W. D.,
wolffian duct.

Fig. 2 Duck embryo (Anas domestica) H.E.C. 2193:3 days, 21 hours. X 42.
t. p., terminal portion of W. D.; y and z, primary and secondary foci of disinte-
gration (z restricted to right side of embryo in this stage).

Fig.3 Turtle embryo (Chrysemys marginata) H.E.C. 1067:6 mm. X 42.
after R. F.Shaner. an.s., primordium of anal sacs (cf. div. c. of chick embryo in
plate 3); x, point of rupture of caudal intestine.

Fig.4 Duck embryo (Anas domestica) H.E.C. 2194:3 days, 21 hours. X 42.
ps. v., ventral half of primitive-streak remnant; z, occluded segment of caudal
intestine.

Fig.5 Chick embryo (Gallus domesticus) H.E.GC. 2071: 3 days, 18 hours.
X 42. (Compare with model of same embryo, fig. 13.) m, marginal sulcus sepa-
rating thin-walled roof from thick-walled sides of cloaca.

168 EDWARD A. BOYDEN

now be defined as representing a persistence of the primitive
streak in the form of a primitive-knot mass. From the cloaca
to the tip of the tail it forms a deeply staining homogeneous mass
differentiating above and below into epithelial structures and
on the sides into the mesenchyma of the tail. The portion
occupying the distal end of the tail is an active tissue giving rise
to the medullary tube, caudal intestine, notochord, and other
caudal tissues. The proximal half, on the other hand, is de-
generating. Some of it may contribute to the mesenchyma of
the tail, but most of it, as indicated by the presence of innumer-
able phagocytes gorged with pycnotic nuclei, is undergoing re-
sorption. This latter portion, representing an excess tissue, is
absent from saurians and mammals, the caudal intestine in these
forms lying close to the inner curvature of the tail. In this
respect the cloaca of the tern (fig. 1) resembles that of lizards and
snakes more than it does that of the gallinaceous birds.

A second process which must be considered in relation to the
formation of the cloacal fenestra is the disintegration of the caudal
intestine. In all reptiles and mammals that I have examined
and in one species of bird embryos (Sterna hirundo, the common
tern) the caudal intestine undergoes reduction in the following
manner. It appears to be pulled out, as if by the elongation of
the tail, so that it tapers uniformly from the newly formed di-
lated portion at the tip of the tail to a slender tube at the oldest
portion—the region adjacent to the cloaca. As the latter por-

3 The details of the process by means of which the primitive streak is segre-
gated in the tail of the embryo will be described in a subsequent paper. At this
time it is sufficient to state that the area described above is derived from that
portion of the primitive streak which is included between the rhomboidal sinus
and the anal plate of a fifteen-somite embryo. In consequence of the folding of
the blastoderm, and of the accompanying overgrowth of the tail, the dorsal por-
tion of the primitive streak, lying under the ectoderm, is folded into the outer
curvature which forms the tip of the tail and thus becomes the tail-bud mass.
The ventral half, lying above the entoderm, and therefore on the inner curvature
of the fold, is tucked under the tail and compressed into the angle between the
anal plate and the caudal intestine.

4 This term of Koelliker’s seems more appropriate than ‘post-anal gut’ intro-
duced by Balfour, since the gut-tract of the tail is an outgrowth of an area which
originally lies anterior to the anal plate. As applied to mammals, the term is still
less appropriate, as the caudal intestine disappears long before the anus is formed.

THE CLOACA IN BIRDS 169

tion becomes more slender the lumen becomes occluded and the
solid strand thus formed soon after ruptures (fig. 3, 2). At
least some of the cells disintegrate and are removed by phagocytes,
but pyenotic nuclei are inconspicuous here as compared with the
abundance of necrotic cells to be found in the degenerating cau-
dal intestine of the chick. This process, which begins at the
cloacal end of the gut, progresses slowly in a craniocaudal direc-
tion until the entire caudal intestine disappears. In duck,
pheasant, and chick embryos, however, the reduction of the
caudal intestine is greatly complicated by the disintegrating
process going on in the primitive-streak mass, asreferred to above,
and by the disintegration of the adjacent cloacal wall, the latter
process resulting in the formation of the cloacal fenestra.

The developmental history of this foramen, which is thus
intimately associated with the removal of the caudal intestine,
is divided into two phases, a period of active disintegration,
beginning at about the 41-somite stage (chick embryo, 2 days,
18 hours), and lasting approximately twelve hours, and a period
of closure, beginning somehere near the 50-somite stage (7-mm.
embryos, of approximately 3 1/3 days), and ending in embryos
of about 9 mm., incubated 3 days and 18 hours. Expressed in
terms of embryonic growth, the first trace of the process appears
just before the wolffian ducts fuse to the cloaca. The final
stage in closure occurs about the time the ultimate somite is
formed (I have found as many as fifty-three) ; that is, before the
resorption of caudal somites begins.

The initial phase, as illustrated by the first text plate (figs.
1 to 5), is based upon two embryos. In consequence of the
great rapidity with which the degenerative process is initiated,
a far greater number of specimens of the same age than were
available would have had to have been sectioned in order to
have provided more than the two stages referred to. For there
is not the slightest indication of the process in an embryo only
one somite younger than the one shown in figure 5, where the
entire area of the cloacal wall which is to be denuded has already
begun to degenerate.

170 EDWARD A. BOYDEN

The first indication of impending disintegration appears in a
duck embryo of forty-five somites (fig. 2). Two paired foci of
degeneration (y and z) are here disclosed in the cloacal wall, one
near the junction of the caudal intestine and cloaca, the other
just anterior to the orifice of the wolffian duct. It is probable
that area y is the first to develop as it is present on both sides of
the cloaca, while area z is present only on the left side. This
specimen, if corroborated by more examples, would seem to
indicate that the degenerative process, which later involves the
caudal intestine, begins in the wall of the cloaca near its junction
with that structure. .

In the next stage (fig. 4, of a duck embryo two somites older),
the two areas on each side have grown together, presenting a
continuous line of degeneration. In addition the lumen of the
caudal intestine has become occluded (fig. 4, x), in the region
which corresponds to the point of rupture in other vertebrates.
This observation is important as indicating the independent ori-
gin of the two processes—the resorption of the caudal intestine
and the formation of a cloacal fenestra—and shows that in the
duck, at least, the caudal intestine becomes detached slightly in
advance of the production of the fenestra. In this specimen,
what remains of the undifferentiated primitive streak (ps. v.)
is appended to the caudal intestine. In the embryo shown in
figure 2, which is younger in other respects, all the primitive
streak has been removed, its former presence being indicated
only by the roughened and irregular ventral margin of the caudal
intestine.

The third stage, illustrated by a chick embryo of forty-one
somites (fig. 5), shows an extension of the area of degeneration
both caudal and cephalad,® and the appearance within this area

5 The cephalic extension contains only scattered phagocytes (represented by
periods in the figure) and does not usually become denuded of epithelium,
although the fenestra has been observed to extend that far in a few cases. If the
cut end of the rectum in figure 5 be examined, it will be noticed that the periods
are limited to a zone of the cloacal wall which is thinner than the adjacent zones.
This area, together with the dorsal wall of the caudal intestine with which it is
continu us and homonymous, represents a persistence of the primitive condition
of the hind-gut which, like the roof of the foregut, is always thin-walled when first

+
¥

THE CLOACA IN BIRDS val

of discontinuous holes where complete resorption of the epithelium
has taken place (fig. 13, from a wax model of the same embryo).
The perforated walls of the cloaca at this period thus simulate
in appearance a fenestrated membrane. Almost immediately,
however, the holes run together, forming a continuous rift along
the cloaca and caudal intestine. In this manner the dorsal wall
of the cloaca becomes detached from the sides and thus isolated
as a trough-shaped structure, is slowly resorbed. Its histologi-
cal appearance will be described later in the paper.

An invasion of the caudal intestine also occurs from another
region in chick embryos and, to a lesser extent, in ducks. This
is an extension of the degeneration process going on in the primi-
tive-streak mass (fig. 5, ps. v.) into the ventral wall of the caudal
intestine, and involves only that part of the intestine which is
adjacent to the primitive streak. Thus, in the undifferentiated
epithelium of the inner curvature of the caudal intestine are
found phagocytes (again represented by periods, fig. 5) which
are coextensive and continuous with the primitive-streak mass,
which is itself undergoing rapid phagocytosis. The occurrence
of these has nothing to do with the imvasion of the caudal
intestine from the cloacal end, except that the two processes
cooperate in destroying that end of the gut.

The resorption of the caudai intestine in birds can now be sum-
marized as follows. In chick embryos the flanks of the caudal
intestine are invaded by a degenerative process originating in the
cloaca, which removes the epithelium before the cavity of the
anal gut can be occluded. In duck embryos the two processes
take place nearly simultaneously, the cloacal invasion slightly
preceding the occlusion of the caudal intestine. Finally, in
terns, the cloacal fenestra is not present at all, and the caudal
intestine undergoes reduction by the method already described
as common to most amniotes.

formed. ‘The side walls are the first to thicken. As development proceeds, the
latter are brought closely together, buckling the flat, thin-walled area into a steep-
pitched roof. But for some time there is an abrupt transition between thick-
and thin-walled portions, and it is along this thin area, and its continuation into
the cloaca, that resorption of epithelium first appears.

172 EDWARD A. BOYDEN

The final stage in the formation of the fenestra, ending the
period of disintegration, is shown in figures 14 and 15, of an
8-mm. embryo of forty-eight somites (3 days and 6 hours). The
entire roof of the cloaca, between the wolffian ducts and the
anal side of the caudal intestine, has been denuded of epithelium,
leaving a considerable gap bounded only by mesenchyma (dash
line, fig. 14). The connection of the cloaca with the caudal
intestine has been lost, and the latter, together with the primi-
tive-streak mass, is now rapidly disintegrating at the ruptured
ends. As a rule, degeneration does not spread any farther ceph-
alad than recorded in figure 5. But occasionally it extends
much farther, and is probably instrumental in producing irreg-
ularities in the dorsal wall, which will be discussed later, in the
section dealing with accessory diverticula.

The cytological changes involved in the formation of the fenes-
tra include the necrosis of the epithelial cells, their removal by
phagocytes, and the reaction of the surrounding mesenchyma to
the denuded area. As seen in ordinary serial sections, the first
step in the disintegration of the flanks of the cloaca is aslight
-oedema of the epithelium which causes the cells to spread apart.
As these become necrotic, the cytoplasm becomes finely granular
and then vesicular and the nuclei pyenotic. At this stage the
epithelium presents a confused histological picture due to the
simultaneous degeneration of so many cells. But almost imme-
diately the cells in regions y and 2g (fig. 2) are resorbed, leaving a
gap in the wall covered only by mesenchyma. At first the
mesenchymal cells appear to congregate about the region, as if
to plug up the opening, and this continues as long as there is an
abundance of necrotic tissue. During the stage when the wall
is a fenestrated membrane the mesenchyma may even invade the
cavity. This is especially true of the caudal intestine which is
eventually replaced by mesenchyma which has grown in through
rifts in the sides and filled the cavity before the walls have been
completely removed.

The most favorable time for appear the cytological changes
is after the gap has been formed on each flank of the cloaca, but
before the roof of the cloaca thus isolated has itself been removed.

THE CLOACA IN BIRDS ie

The degenerating epithelial cells bordering the gap may then be
studied in less crowded condition. Such a picture is presented
in figure 19—an obliquely frontal section passing through the
fenestrated area at right angles to the back lines of the cloaca;
that is, in a plane cutting the allantoic duct lengthwise. In
this figure the following features should be noted: the isolated
roof of the cloaca, rows of necrotic epithelial cells on either flank,
the concentration of mesenchyma about the gap on either side,
and the rounded margins of the epithelium conspicuous by their
failure to regenerate. In the epithelium bordering the gap are
occasional pycnotic nuclei, and here and there a phagocyte,
indicating a slow resorption in contrast to the sudden removal
characteristic of initial stages. When the degenerative process
slows down and finally comes to an end, a single large foramen
is left in the dorsal wall of the cloaca extending from behind the
level of the wolffian duct to the site of the caudal intestine, hav-
ing a lenticular shape when viewed from below (fig. 15). As
seen in microscopic section (fig. 20) the epithelium of the roof
of the cloaca has been entirely removed, leaving in its place a line
of mesenchymal cells which have flattened out into a surface
layer as if under compression by the fluid in the cavity, in a
manner recalling the formation of the false epithelium which
lines the joint cavities.

Even before degeneration stops, however, the process of closure
setsin. This consists of a fusion of the epithelial margins of the
gap beginning at the caudal angle of the aperture, so that in
the space of another twelve hours, only a slender cleft remains at
the anterior end of what was once a big fenestra (fig. 23, fen.).
This process of closure seems to be aided if not caused by
a progressive approximation of the sides of the cloaca, beginning
at the anal plate, which results in the fusion of opposite walls and
the formation of a urodaeal membrane. Figure 21, of a cross-
section of the fenestra in the last stage of closure, shows that even
to the end of closure no regeneration of the cloacal lips has taken
place, but that rather the free margins of the walls have been
pushed down into the mesenchymal cavity, as if by lateral com-
pression exerted upon the side of the cloaca. By the middle of

174 EDWARD A. BOYDEN

the fourth day of incubation all signs of the cloacal fenestra
have disappeared, and its site cannot be accurately located ex-
cept in such general terms as lying between the accessory bursa
and the urodaeal sinus.

In concluding this chapter one may say that the most conspicu-
ous feature of the entire process is the rapidity with which it
takes place—both the sudden appearance of a gap and the rapid
closure of it—all occurring within a period of twenty-four hours.
Although the evidence presented would lead one to infer that
the disintegration of the cloacal wall precedes the reduction of
the caudal intestine, and is thereby independent of it, and calls
for a separate explanation, it is still possible that the cloacal
fenestra represents a modification or extension of the process by
which the caudal intestine is reduced in other vertebrates. Any

attempt, however, to explain the significance of this foramen in”
the domestic fowl, duck, and pheasant, must take into account.

an equally peculiar feature, likewise found only in birds with a
fenestra, namely, the undue persistence of the primitive streak
in the proximal end of the tail. It is well known that the tail in
modern birds, and of fowls in particular, is shorter than in the
‘Archaeornithes. It is conceivable that the degenerating primi-
tive-streak mass in the tail of the chick embryo represents a
persistence of material once utilized in tail-building but now
superfluous. It would also seem, from a comparison of the clo-
acas in the first text plate, that the persistence of this indiffer-
ent tissue hasdelayed the differentiation of the caudal intestine and
perhaps of the whole tail itself. For figure 5 represents a chick
embryo in which the ventral wall of the caudal intestine has
not been differentiated into an epithelium, but is still continuous
-with the primitive streak throughout its length. Yet that chick
is older in other respects than the tern embryo of ‘figure 1, as
evidenced by the lesser number of somites in the chick, and by
its greater maturity of form. If it be granted that the devel-
opment of the caudal intestine in the chick has been retarded by
the persistence of the primitive-streak mass, it is not inconceiv-

Y
é

THE CLOACA IN BIRDS 175

able that the development of the corresponding region in the
adjacent cloacal wall has likewise been interfered with, and that
when reduction of the caudal intestine does occur, both of these
areas are subjected to a retrograde process more rapid and exten-
sive than obtains in other vertebrates.

DEVELOPMENT OF THE UROGENITAL APPARATUS

Anomalies arising in connection with the wolffian ducts

About the time that the primary excretory ducts reach the
level of the cloaca in their downgrowth from the pronephros,
an eruption of diverticula appears on each flank of the cloaca
opposite the distal portion of the ducts. Since these outpocket-
ings of the cloaca seem to develop in response to the presence of
the wolffian ducts, and later fuse with them, I have named them
complemental diverticula. A surface view of this stage, such as
is shown in figure 13 of a 41-somite chick embryo (62 hours),
reveals the presence of two groups of diverticula—a circlet of
five or six small ones opposite the terminal portion of the duct,
and a single larger one farther up on the shaft, as broad as the
whole field of smaller ones. In this embryo the duct of the left
side has not fused with the cloaca, although fusion on the right
side has taken place. In a 40-somite embryo neither duct.has
fused. My observations would therefore differ somewhat in de-
tail from the statement of Lillie that the wolffian duct ‘‘reaches
the cloaca (with which it unites) about the 3l-som. stage’’ and
that ‘‘at about the sixtieth hour the ends of the ducts (described
in the preceding sentence as solid) fuse with broad lateral diver-
ticula of the cloaca, and the lumen extends backwards until the
duct becomes viable (?) all the way into the cloaca (at about 72
hours, 35 somite stage).’”’ For a frontal section (fig. 6) of the
cloaca shown in figure 13, at the place where the left wolffian
duct makes the nearest approach, shows that the duct has not
yet fused with the cloaca, that its terminal portion is patent,
and that the mesial wall of the duct is thinning out in anticipa-
tion of fusion. The section through the left side happens to

176 EDWARD A. BOYDEN

pass through three diverticula, the broad one (a), and two smaller
ones (b and c, members of the terminal circlet of diverticula).
The arrow indicates that the duct in sections higher up would
reach as far as the point c. In subsequent stages the mesial
wall of the duct would fuse with the cloacal diverticula forming

WI

SES
\
\
Fig. 6 | ;

TEXT PLATE ILLUSTRATING ANOMALIES OF THE WOLFFIAN DUCT

Fig.6 Chick, H.E.C. 2071 (section 661):2 days, 18 hours. X77. a, proximal
complemental diverticulum; 6 and c, distal complemental diverticulum; arrow
indicates extent of wolffian duct in other sections. Note thinning out of mesial
wall of duct in preparation for fusion with cloaca.

Fig.7 Duck, H.E.C. 2194 (section 680): 3 days, 21 hours. X 77. mes.,
mesenchyma interposed between distal and proximal attachments of duct.

Fig.8 Chick, H.E.C.2073:2 days,2lhours. X77. x, plate formed by fusion
of mesial wall of W. D. with complemental diverticula of cloaca; arrow shows
where plate has been ruptured, through distal diverticulum. 2

Fig.9 Chick, H.E.C.2072:2 days,22 hours. X77. Arrow shows where plate
has been ruptured through proximal diverticulum.

Fig. 10 Model of duck embryo, H.E.C. 2197: 4 days, 8 hours. X 40. all.,
allantois; an. pl., cloacal membrane; c.7., caudal intestine; cy., epithelial cysts of
unknown origin; fen., fenestra; t. p., terminal portion of W. D.; ur-, primordium of
ureter; W.D., wolffian duct.

Fig. 11 Model of duck embryo, H.E.C. 2195: 4 days, 8 hours. X 40. div.,
aberrant complemental diverticulum.

THE CLOACA IN BIRDS ie

a continuous plate (figs. 8 and 9, x) from a toc. In some cases
the plate ruptures first through the distal diverticulum (see
arrow in fig. 8); in others at first through the proximal one (fig.
9). But in all chicks of older stages that I have examined, the
plate is resorbed, leaving a single large opening from a toc. It
is probable that phagocytes aid in this resorption, as I have found
them within the thin plate as soon as the duct has joined the
cloaca. As development proceeds, the lateral walls of the cloaca
beginning with the anal plate gradually come together, forming
a solid membrane comparable to the urethral plate of mammals,
so that finally the opening of the wolffian duct becomes restricted
to the middle of the cloaca at the level a of figure 6 (cf. figs. 14 and
16). Not all of the complemental diverticula, however, fuse
with the ducts. Some of them, no doubt, are soon suppressed.
Others of them persist for a longer or shorter time, growing out
as accessory diverticula (figs. 11, 22, 24, and 32, div.).

The most interesting anomalies occur in duck embryos, and
are due to the excessive length of the wolffian duct, which nor-
mally grows down to the very end of the cloaca (fig. 7). In one
case observed, only the proximal portion of the duct had fused
with the cloaca, the terminal portion growing out as an aberrant
diverticulum (fig. 10, ¢.p., left). In other cases both terminal
and proximal portions fuse, but not continuously, so that an
area of mesenchyma is left between the two attachments (fig. 7,
mes.). If, then, the basal ends of the ducts begin to grow,
a ring-shaped (fig. 10, ¢.p., right) or a U-shaped (fig. 11, t.p.,
left) attachment of the ducts is formed, opening into the cloaca
at two points, representing the original points of fusion. A
similar anomaly has been found in a chick embryo (H.E.C.
99), and it would seem almost certain that a larger number of
specimens would show many indications of aberrance resulting
from the fusion of the wolffian duct to the complemental divertic-
ula. The further changes in the form of the wolffian ducts and
their incorporation into the wall of the cloaca will be considered
in the next chapter.

178 EDWARD A. BOYDEN
Formation of the wrodaeal sinus

In discussing the origin of urinary bladders Felix defines four
main types: 1) mesodermal bladders, arising from the fusion or
dilation of the caudal ends of the wolffian duct; 2 and 3) dorsal
and ventral cloacogenic bladders, outgrowths or dilations of the
dorsal and ventral walls of the cloaca, respectively, and, 4)
allantoidogenic bladders formed by the retention of the proxi-
mal end of the allantois. The first type in its pure form is real-
ized only in selachians, the second type only in amphibians,
both groups being devoid of an allantois. The bladders of all
other vertebrates, according to Felix, are of mixed origin. When
we examine birds, it appears that they are the only class among
amniotes without one or more bladders, yet curiously enough,
reptiles, from which birds have descended, constitute the class
with the greatest number and diversity of bladders. Thus,
according to Felix, lizards derive their bladders from three sources,
dorsocloacogenic, allantoidogenic and mesodermal; and in
turtles the bladder is formed from dorsocloacogenic, ventro-
cloacogenic, allantoidogenic, and mesodermal origins (Keibel
and Mall, II, p. 869). It would be strange, then, if the bird did
not exhibit some traces of bladder formation in its ontogeny,
and such, in fact, may be found. The most conspicuous of
these is the intra-embryonic expansion of the allantois shown in
figure 39. It is almost identical at this stage with the primor-
dium which develops into the ventral bladder in most reptiles.
But it is completely resorbed in adult birds.

The other structure in bird embryos which recalls the reptil-
ian bladders (this time those of dorsocloacogenic and mesoder-
mal origin) is the wrodaeal sinus, a name which I have applied to
the cavity of the urodaeum at its maximum extent (figs. 40 and
41 urod.). Minot in 1900 called attention to the peculiar
relations of this cavity as follows: ‘‘From the closure of the
intestinal opening by the entoderm (occluded rectum), and of
the anal opening by the anal plate (meaning urodaeal membrane),
there is left a clear passage from the wolffian duct across (to)
the opening of the allantois.’”’ And he quotes the suggestion

ox

THE CLOACA IN BIRDS 179

offered by G. H. Parker that “the physiological purpose of this
arrangement is to secure the transmission of the excretion from
the embryonic kidney to the allantois, and to prevent the escape
of the excretion, either into the intestine or into the amniotic
cavity, where it might prove injurious to the embryo.” ‘That
the urodaeal sinus is a mechanism inherited directly from rep-
tiles was revealed two years later by the comparative studies of
Fleischmann and his students on the cloaca and phallus of liz-
ards, snakes, turtles, birds, and mammals. He notes that in
the Sauropsida the urodaeum is divided into two poriions, a
distended oral portion always in relation to the wolffian ducts,
and an elongated caudal portion which forms an open passage-
way (even in young embryos) to the anus. The shutting off of
the urodaeal sinus from below in birds is due to the fact that the
second half of the urodaeum never elongates, but remains short
and impervious through the formation of a urodaeal membrane.

While the posterior portion of the urodaeum becomes elongated
and subject to great modification in various reptiles, the anterior
chamber (urodaeal Kammer of Unterhéssel) is always associated
with bladder formation. It becomes chiefly dilated in a dorso-
lateral direction, so that the entire cavity and associated meso-
dermal ducts assume the appeance of a dorsal bladder (cf. Fleisch-
mann, Taf. VIII, figs. 1, 2 and 4). This striking feature appears
temporarily in bird embryos as the urodaeal sinus, and is as con-
vincing a repetition of reptilian ancestry as the allantoic bladder
previously referred to in figure 39. But since it was studied
chiefly in older embryos, and then largely by means of sagittal
sections, its extent and composition was not fully appreciated
even by Fleischmann.

As seen in figures 40 and 41, the urodaeal sinus (wrod.) is a greatly
inflated segment of the cloaca, placed athwart the main axis of
the hind-gut, between the occluded rectum and the urodaeal
membrane. Its lumen from front to back is reduced to the size
of a fissure, but is greatly expanded laterally and dorsoventrally,
extending from the wolffian duct of one side to that of the other
and from the dorsal side of the cloaca to the allantois. Although
existing as a single structure at this stage, it has been formed

180 EDWARD A. BOYDEN

by the confluence of three originally separate elements. The
first of these to appear is the median diverticulum designated
as diverticulum c in the reconstructions shown in plate 3. It
arises as early as the beginning of the fourth day and main-
tains its identity as a distinct and conspicuous feature of the
cloaca as late as the seventh day, at which time it is incorporated
in the urodaeal sinus. This structure has been figured in descrip-
tions of the avian cloaca as far back, at least, as the work of
Bornhaupt (’67). But I question whether its existence as a sepa-
rate rounded diverticulum has ever been appreciated. Pomayer,
in the Fleischmann series, labeled it “‘Urogenitaltasche” in a
sagittal section of a duck, giving it the same designation as the
paired urogenital pockets of the snake, Tropidonotus, which are
dilated outpocketings on the dorsal wall of the cloaca into which
the wolffian ducts empty. A median diverticulum occurs in the
same place (as diverticulum c) in the turtle embryos modeled
by R. F. Shaner (fig. 3, an. s.), and has been interpreted by
that author as the primordium from which the respiratory sacs
(bursae anales) of turtles develop. In view of its position between
the two wolffian ducts in both chicks and turtles, it seems
not improbable that diverticulum c represents the dorsal out-
pocketing of the cloaca of reptiles from which the wolffian
ducts have shifted in course of their migration to the allantois.

The second and third components of the urodaeal sinus arise
more or less together. As seen in figures 14 and 6, the wolffian
ducts, when they first reach the level of the cloaca, fuse to the
cloaca along a broad area extending from the caudal margin to
near the allantois (a to c). The fusion at ¢ approximates the
primary position of the excretory ducts in lower vertebrates.
In consequence, however, of the fusion of the two side walls of
the cloaca, beginning with the anal plate, to form the urodaeal
membrane, the outlet of the wolffian ducts at c and 6 in figure
6 is suppressed. The broad complemental diverticulum (fig.
6, a) thus becomes the main channel, and in course of develop-
ment is enlarged into a wing-like expansion of the cloaca connect-
ing the wolffian duct with the neck of the allantois (fig. 16).
Meanwhile the segment of the wolffian duct between the orifice

THE CLOACA IN BIRDS 181

of the ureter and the cloaca begins to develop irregular enlarge-
ments sometimes suggesting diverticula (fig. 17), which eventually
result in the widening of that segment. By the eighth day the
distended ends of the wolffian ducts have been taken up in the
urodaeal sinus as far as the origin of the ureters, the latter ducts
in this process rotating from the dorsal to the mesial border of
the wolffian duct. From this period on, the original components
lose their identity in the sinus. In the adult the depth of this
cavity is greatly reduced, the whole forming a shallow transverse
segment, the definitive urodaeum, the latter being separated
from the coprodaeum by the urorectal fold of Retterer and from
the proctodaeum by the uro-anal fold. The position of these
folds in the embryo is evident as early as the beginning of the
fourth day of incubation.

Another interesting feature of the urogenital apparatus which
occurs at this time is the constriction of the metanephric pelvis
at its lower third into a narrow isthmus (fig. 39). This was fig-
ured by Schreiner (’02), who noted its relation to the umbilical
arteries. Asis well known, the adult kidney of birdsisconstricted
into three lobes. The cause of the upper constriction is yet to
be determined; the lower constriction is accounted for by the
mechanical obstruction offered by the umbilical arteries. The
developing kidneys of the pig, as shown by Lewis and Papez,
are similarly caught in the bifurcation of these vessels, but in-
stead of becoming notched as in the bird, they escape by moving
upward, sometimes, however, being brought so near together as
to fuse from side to side, forming a ‘horseshoe kidney.’

In closing this chapter I wish to call attention to the changes
which have been taking place in the terminal segment of the
intestine. In figures 35 and 40 its lumen is shown to be occluded
for some distance, the solid tube thus formed joining the uro-
daeal sinus by a thin linear attachment. By the fifteenth day
the cavity of the coprodaeum has been reéstablished and consid-
erably distended except at the solid linear attachment. This
greatly dilated chamber at the end of the intestine (fig. 41,
copr.) is unquestionably homologous with the lower end
of the rectum of the human foetus, as figured by Johnson (14).

182 EDWARD A. BOYDEN

This includes a rectal ampulla passing below into a plicated
‘zona columnaris.’ In the chick embryo it is bounded above by
a single transverse plica and below by the urorectal fold already
mentioned. Since this ampulla functions as a part of the cloaca
in the adult bird, being the chamber in which both fecal matter
and urine are retained, it seems better to keep the name copro-
daeum, which Gadow applied to the most anterior of the three
divisions of the cloaca.

DEVELOPMENT OF THE BURSA OF FABRICIUS AND ASSOCIATED
DIVERTICULA

The primordium of the bursa is usually described as a swelling
in the caudal wall of the cloaca, caused by the coalescence of
vacuoles arising within the urodaeal membrane during the fifth
and sixth days of incubation (figs. 31 and 18, bursa). While
modeling earlier stages of the cloaca in relation to the develop-
ment of the fenestra, I was much surprised to find that all chick
embryos which had been incubated about four days showed a
conspicuous diverticulum at the site of the caudal end of the
cloacal fenestra, measured by its greatest extent (figs. 24 and 27,
a; cf. figs. 16 and 18). The picture was further complicated by
the occurrence, in several cases, of a second diverticulum (fig.
24, b), arising as an outpocketing of the cloaca at the site of
the cephalic end of the fenestra. Furthermore, diverticulum a,
while originally developing as an invagination of the cloaca, soon
became solid, then vacuolated, in continuity with the vacuoles in
the developing urodaeal membrane (fig. 28, a), and then, by
fusion of vacuoles, appeared to develop into the bursa itself
(fig. 30, bursa). In view of these facts, it seemed not improbable
that diverticulum a represented an earlier and more significant
stage in the origin of the bursa than had hitherto been reported—
a stage which had been overlooked because the cloaca had never
been modeled during this period of its growth. This interpreta-
tion, if true, would be of importance as bringing the origin of the
organ into line with other derivatives of the gut. For it would
show that it originated as an invagination of the entodermal tube,
thus removing one more difficulty in the interpretation of an

THE CLOACA IN BIRDS 183

organ which has been a bone of contention among anatomists
since its discovery by Fabricius. The chief obstacle to this
conclusion, however, arose from the examination of a single
specimen pictured in figure 29. In this figure diverticulum a
seemed farther removed from the anal plate than in other speci-
mens, thereby leaving a vacuolated area between it and the anal
plate (labeled bursa in the drawing) which might well develop
into the bursa of figure 30, there recognized as the definitive
bursa by the coalescence of the vacuoles. To solve this difficulty
it became necessary to collect a series of graded embryos of other
species of birds. Subsequent reconstruction of domestic duck
and pheasant embryos left the matter still more confused, as in
these forms the diverticula were present and similar to those in
the chick, but less pronounced. Finally, an examination of
tern embryos, birds some distance removed from the gallina-
ceous tribe, brought the desired results. In these forms, as can
be seen in figure 36 to 38 and reconstructions of earlier stages,
no diverticula are developed at all, and the bursa arises directly
from the region adjoining the anal plate, as a thickening of
epithelium in continuity with that plate and restricted to the
territory lying between it and the site of the caudal intestine
(fig. 1). A reexamination of chick embryos in the light of these
facts has led to the following conclusions. The bursa of Fabri-
cius in the chick begins soon after the rupture of the caudal
intestine, as early as the beginning of the fifth day, as a prolif-
eration of entodermal epithelium on the caudal border of the
cloaca adjoining the anal plate (fig. 26, bursa), but it does not
develop from the epithelial elements which originally belonged
to the caudal intestine, as maintained by Stieda. As the two
walls of the cloaca, beginning at the anal plate, progressively
fuse to form the urodaeal membrane, vacuoles appear in the
solid plate thus formed (figs. 27, 28, and 29, bursa). Those on
the free border adjoining the anal plate coalesce and distend the
cloaca, forming the definitive bursa of Fabricius (fig. 30, bursa).
Previous to these events, however, a diverticulum may appear at
each end of the area marking the site of the cloacal fenestra.
The caudal diverticulum (a) is always present in chick embryos,

184 EDWARD A. BOYDEN

where it is associated with the bursa of Fabricius (figs. 33 and
34). The other diverticulum (6), when present, becomes
associated with the urodaeal sinus (fig. 32, div. c). Both of them
are probably to be regarded as irregularities produced at either
end of the fenestra by the removal of intervening epithelium.
They are present only in those birds which exhibit a fenestra,
and are most conspicuous in that species which has the largest
fenestra—the domestic fowl. The regularity with which di-
verticulum a appears maty be explained by the fact that the
posterior end of the fenestra is always larger, and that diverti-
culum a, when first formed, arises from the prominence to which
the primitive streak of earlier stages was attached (cf. figs. 21
and 23).

The later stages of development, which have been partly
described by previous authors on the basis of sagittal sections,
are shown in figures 34 and 39, 35 and 40, and 41. These models
illustrate the development of the bursa up to the period of his-
tological differentiation. The successive steps leading to this
period are: 1) the continued outgrowth of the bursa and sim-
ultaneous enlargement of its cavity through further coalescence
of vacuoles; 2) the projection of the anal sinus (proctodaeum)
in a ventrodorsal direction across the flanks of the urodaeum on
its way to connect with the bursa (cf. figs. 18 and 39); 3) the
breaking through of the thin plate separating the cavity of the
bursa from the proctodaeum (ef. figs. 34 and 35), and, lastly
(fig. 41), the differentiation into three parts of the passage-way
thus made continuous from anus to the end of the bursa. At
this stage (eleventh day) this passage-way is still separated from
the rest of the cloaca by the urodaeal membrane, which does not
rupture until after the seventeenth day (Gasser). As seen in
figure 41, the first of its three parts, the proctodaeum of ecto-
dermal origin, has assumed the shape of a compressed chamber
with broad flange-like expansions. By the fifteenth day ecto-
dermal glands have begun to differentiate around its circumfer-
ence. The second and third parts, of entodermal origin, have
developed, respectively, into a short bursal stalk and a greatly
expanded but plicated sac, the bursa itself (fig. 41). The cavity

ae ae

eee Db wo ek

THE CLOACA IN BIRDS 185

of the latter is subdivided by longitudinal plicae into eleven
(or twelve) grooved chambers. A cross-section of the bursa
during the fifteenth day (fig. 12) shows that in the interval be-
tween the eleventh and fifteenth days some of the primary plicae
have cleft the central cavity deeper than others, so that the
eleven primary cavities have become tributary to six or seven
secondary channels, opening into the main cavity after the
manner that minor and major calyces open into the pelvis of
the kidney.

Fig.12 Transverse section of a model of a 55-mm. chick embryo, H.E.C.
1968:14 daysand5 hours. X 28. bl. v., blood vessel; cav., cavity of bursa; cor.,
cortex of follicle, derived from tunica propria; med., medulla of follicle, derived
from epithelium; musc., muscularis; ¢t. p., tunica propria.

Histogenesis begins with the appearance of the primary plicae
and ends in the formation of spherical masses of lymphoid tissue
(the ‘follicles’ of Stannius). Each follicle consists of a cortex
and a medulla, the medullae or cores of the follicles (the ‘¥olli-
kelkeime’ of Stieda) being the first to appear. These grow out
into the tunica propria as solid buds of epithelium which soon
become clothed peripherally with a cortical layer derived from
the subjacent connective tissue (fig. 12, cor. and med.). In
the course of development the follicles grow larger and larger
until they meet, the resulting pressure molding them into a
polyhedral shape. The walls of the bursa thus become greatly
thickened, resembling somewhat in gross appearance the walls

186 EDWARD A. BOYDEN

of the proventriculus (glandular stomach of birds) to which the
bursa was compared in 1829 by Berthold. In the region next
to the stalk, according to Schumacher (’03), the follicles are
neither so thick nor so sharply limited, but look more like a
diffuse infiltration of tunica propria with lymphocytes. To these
finger-like processes, which in my model of the fourteen-day
chick are restricted to the dorsal wall of the bursa where it joins
the stalk, Schumacher has applied the term mucosal villi.

The nature of the epithelial transformation has received several
interpretations. Wenckebach (’88) and Schumacher (’03) main-
tain that the entodermal epithelium constituting the medulla of
each follicle is differentiated directly into lymphoid tissue, and
that this process is followed by a differentiation of the mesenchy-
mal cortex into a similar tissue, the border-line between the two
layers becoming ill-defined in later stages. Retterer, in his
latest paper (713), extends the activity of the epithelium still
further, stating that ‘‘the cortex of the follicles of the bursa is
likewise of epithelial origin.”” The most comprehensive account,
however, is that of Jolly (15), who based his conclusions not
merely upon histogenesis, but also upon the involution of the
organ (both natural and induced) and upon examination of
tissues in vitro. Beginning with the eleventh day of incubation,
he finds numerous amoeboid cells, formed directly from the
mesenchymal network, accumulating in the vicinity of the epithe-
lial buds. These they soon invade, the most active phase of
penetration occurring between the fourteenth and eighteenth
days. Although at first the epithelial cells give way to the new
arrivals, by becoming detached from one another and in some
cases by even degenerating, the majority of them, he maintains,
enter upon a symbiotic relation with the invaders by means of
which both cell strains continue to divide actively, the amoeboid
cells giving rise to large numbers of small lymphocytes, the
epithelial cells forming a reticular network within which the
lymphocytes reside. Simultaneously the cortex becomes differ-
entiated into a highly vascularized lymphoid tissue.

In involution the order of events is reversed; the lymphocytes
in the medulla die and the epithelial cells close their ranks, tend-

THE CLOACA IN BIRDS 187

ing to reconstitute themselves into a compact epithelial bud—
a process which Jolly has compared to the production of Hassal’s
corpuscles in the thymus. As involution continues the follicles
separate from the epithelium and become replaced by fibrous
tissue, the whole process taking place progressively from apex
to base of the bursa in such a way that a gradual but rapid dimi-
nution of volume and weight ensues. During the eighth month
the bursa loses all possibility of functioning, and in the course
of the next two months becomes reduced to a thin-walled cyst,
still opening into the cloaca at its posterior end, but so completely
fused to the aponeurosis of the rectum that it can be detected
only by careful dissection. In this condition it may persist
until old age. Only in the Ratitae, according to Forbes, does
it remain as an undiminished organ throughout life where, by
virtue of its broad opening into the proctodaeum, it becomes a
repository for the urine. In these birds, according to Gadow,
micturition and defecation are separate processes, whereas in
most other birds the urine backs up into the coprodaeum and
there mixes with the faeces until evacuated.

The following table, arranged from data submitted by Jolly,
is introduced to summarize the growth and involution of the
bursa in the fowl:

Ane Length Weight

mm. grams

5B Gi 3) LSU ae eae Ue ek Sailer Re A ga a 5 0.05
Ilse Wael. sine can Sata Pek REET Ee io eeD ree ea ie 10 0.50
2A WYNN Des SOS hae SSE ROLE See e eT TLE 15-18 0.50-1.0
SHoT COVE AN ANSE ets 485 oe Spe ca oe as tL EG ae ee OR VT 20-25 1 6
MERILOREUHS Meee ee ee ee ees eae ine § 30 3.0 (gn of body)
A AN OIG Se Ee ENS eee ch ie Sea 2.51
OEM On Hs ey 5. cieypaeeryseneitus sires Set titerons Tete tate ooh 0.97
GET OMG S secs cee eS ts aie ote oto es eee rors 0.22
Fy SEUCG TTI IE stag Utes A pt SR a iE 10-20 0.26

Lem On hh ewes s wae GPA eokede fo eles fh ee ees 0.12

The function of the bursa has never been satisfactorily ex-
plained. Jolly’s description of the haematopoietic foci of the
bursa, from which he derives not merely lymphocytes, but also
red corpuscles and granular leucocytes, has added something to
our knowledge of its activity, but, as he well recognized, this

188 EDWARD A. BOYDEN

function is not peculiar to the bursa, but is an attribute common
to the mesenchyma of certain other organs. He does, however,
propose a specific function when he suggests that the bursa
contributes substances to the organism which bear a causal re-
lation to the inception of sexual maturity. He bases this theory
on two facts: 1) that the maximum development of the bursa
is attained at the time when spermatogenesis is just getting under
way; 2) that involution of the bursa corresponds exactly with the
appearance of sexual maturity, as measured by the sudden in-
crease of testicular weight and the appearance of ripe sperma-
tozoa. Before accepting this theory, however, one would like
to know to what extent the precocious involution, which Jolly
produced in the bursa by means of the x-ray, affected the differ-
entiation of the testis. ‘That some such line of experimentation
as this would be profitable seems almost certain when we consider
the history of such organs as the thymus. For it is far from in-
conceivable that the bursa may also be a glandular organ in
process of transformation into an endocrine gland, if it has not
already arrived at that estate.

The phylogenetic interpretation of the bursa is equally obscure.
An extensive number of investigators distributed over three
centuries have tried to solve this problem and during this period

have proposed numerous hypotheses, all of which have been .

rejected (see Retterer, 13 b, for list). Forbes, after examining
the bursae of over ninety species of birds and covering the litera-
ture, came to the conclusion that the bursa was a glandular
outgrowth of birds sui generis. Wenckebach limited the problem
by establishing the entodermal origin of the bursa, thus making
obligatory the origin of homologous structures (with which it
is to be compared) from the dorsal wall of the vertebrate cloaca.
Its origin has been still further limited by this paper to the area
between the cloacal end of the caudal intestine and the anal
plate.

These limitations render untenable the hypothesis put forth
by Stieda (80) that ‘“‘the bursa develops from the epithelial
elements which originally belong to the caudal intestine.” Equal-

ly untenable is the modification of this theory, presented by

a

THE CLOACA IN BIRDS 189

Fleischmann (’02).6 Recently Stieda’s point of view has been
revived again, this time by Jolly (15), who has made it a basis
for the theory that the bursa represents a recrudescence of the
cloacal end of the ruptured caudal intestine.

“The first anlage of the bursa,” he writes in his conclusion,”
occupies exactly the situation of the post-anal intestine and it is
orientated like it; it may be said, even, that the anlage blends
with what remains of the post-anal intestine. One may consider
that the bursa represents the remainder of the caudal intestine
which rises up again posteriorly and, turned toward the head,
undergoes a further development under the form of a true cloacal
caecum, in the walls of which lymphoid tissue develops.”’

In refutation of this.theory, new evidence, presented in the
first section of this paper, shows that the entire region of junction
between caudal intestine and cloaca, together with the adjacent
wall of the latter, has been removed by the process which forms
the cloacal fenestra. There is, therefore, nothing left of this
end of the caudal intestine which Jolly assumes to be present
and which he describes as growing out, in an unusual direction,
to form the bursa. Furthermore, even after the closure of the
fenestra, the bursa does not arise at the site of the former caudal
intestine, but on the anal side of it, beyond diverticulum a (figs.
25 to 33).

Another theory, presented during the last ten years, is that of
Osawa (711), who has revived the hypothesis of Martin St. Ange
(56). He believes that the bursa is homologous with the pros-
tate gland even though the latter is well developed in the male
only. Osawa bases his conclusions on the ground that the “‘ bursa
occupies the place where the ureter and ductus deferens dis-
charge themselves, and its follicles are laid out after the manner
of glands.” In refutation of this view, it may be stated that the

§ In a foot-note to his paper (p. 58) Fleischmann suggests that ‘‘the caudal
process of the primitive urodaeum of mammals, which now bears the perplexing
name caudal intestine, is comparable morphogenetically with the bursa of Fabri-
cius.”’ This conjecture has recently called forth the following rejoinder from
Keibel (’21): ‘‘The caudal intestine of birds has not the slightest thing to do
with the bursa of Fabricius.”’

190 EDWARD A. BOYDEN

point of origin of the group of glandular outgrowths that con-
stitute the prostate gland is rather remote, embryologically,
from that of the bursa; also that the prostate develops much
later and is radically different in its histological nature. Physi-
ologically it becomes functional with sexual maturity, at the
time when, as Jolly has shown, the bursa degenerates.

The only other vertebrate structures thus far proposed, which
in any way meet the requirements of the homology, are the anal
sacs (bursae anales) of turtles. Gadow, in the Cambridge
Natural History Series, 1909, describes these organs in the adult
as highly vascularized, thin-walled sacs which are incessantly
filled and emptied with water through the vent, and act as im-
portant respiratory organs. Forbes, in. 1877, objected to the
comparison of these sacs with the bursa of Fabricius on the ground
that they were paired, lateral structures. Wenckebach also
saw this objection, but considered that the anal sacs were the
only diverticula which in any way could be compared in point of
origin with the bursa, and, in view of the almost total ignorance
regarding the embryology of the sacs, held that the objections
to the comparison should not be conclusive. During the last
year a graded series of models of the turtle cloaca have been
made in this laboratory by R. F. Shaner as a part of an anatomi-
cal study of the 9.5-mm. Chrysemys embryo. As a result of
this study he is of the opinion that the anal sacs arise from a
single median diverticulum (fig. 3, an. s.).. Through the courtesy
of Doctor Shaner, I have had the pleasure of studying the models
upon which his paper is based and concur in his opinion. Another
feature which at first seemed to favor the comparison between
the bursa and the anal sacs is the striking similarity of the proc-
ess by means of which the outlet of each diverticulum is taken
over by the proctodaeum. In each case lateral expansions of
the proctodaeum (fig. 39) grow down across the flanks of the
cloaca until they establish communication with either the bursa
or the anal sacs. But the description of the saurian cloacas
in the Fleischmann series seems to indicate that this invasion
of some point of the urodaeum by the lateral proctadaeal in-
vagination is not restricted to reptiles equipped with anal sacs,

THE CLOACA IN BIRDS 191

but occurs in most other reptiles. Another objection to this
homology is based upon the fact that the anal sacs arise on the
cephalic rather than on the anal side of the caudal intestine.
They are thus more nearly comparable to diverticulum c, which
unquestionably represents the urodaeal Kammer or dorsal
bladder of the saurian cloaca, than to the bursa of Fabricius.
In Unterhéssel’s account of the saurian cloaca another divertic-
ulum is represented which, as a possible homologue of the bursa,
seems much more promising. This is an invagination of the
dorsal wall of the cloaca, defined by Unterhdssel as lying at the
junction of the urodaeum and the proctodaeum. It is figured
in models of late embryonic stages of three different species,
and would seem to be a modification of the same structures.
The first is a vaulted portion of the roof of the urodaeum of the
lizard Platydactylus guttatus (Taf. VIII, fig. 1, st). The second
is a comb-shaped diverticulum occupying the. same position in
the cloaca of the snake Anguis fragilis (Taf. VIII, fig. 2, not
labeled). The third consists of a pair of dorsal diverticula lying
behind the urodaeal chamber and described as outpocketings
of the proctodaeum in the snake Tropidonotus natrix (Taf. VIII,
fig. 4, s). But it will be remembered that the bursa for a long
time was described as an outgrowth of the proctodaeum, and
the author in this case admits the lack of younger stages. From
an examination of the account of the saurian cloaca I am con-
vinced that the key to the homology of the bursa of Fabricius
lies in the study of the reptilian cloaca, and am optimistic enough
to believe that such a careful study of the younger stages of the
reptile cloaca as Fleischmann and his students have made of
older stages will bring the desired results. The comparison
which Schumaker has lately made with the tonsiloid tissue dis-
covered by Keibel in the cloaca of the mammal Echidna does not
seem to meet the problem. At best it can only be considered a
vestige of a reptilian prototype, and to reptiles we must again
direct our attention for interpretation of the bursa of Fabricius.

192 EDWARD A. BOYDEN

SUMMARY

This paper represents a review of the development of the
cloaca in bird embryos from the third to the fifteenth day of
incubation. It is based on the study of a large number of chick
embryos supplemented by observations on three other species
of birds. The most striking feature to be recorded is the regular
occurrence of a temporary fenestra in the wall. of the cloaca,
caused by the disintegration of a definitely localized area of
epithelium and its subsequent removal by phagocytes, following
which the contents of the cloaca are left in contact with the
mesenchyma for a period of nearly twenty-four hours. It is
of interest not merely because it furnishes the only instance in
the differentiation of a hollow organ in which a gap occurs in
the epithelial wall as a normal and constant feature of develop-
ment, but also because it enables us, by virtue of the landmarks
it establishes, to determine for the first time the exact point of
origin of the bursa of Fabricius.

The second part of this paper deals with the formation of a
temporary sinus, placed athwart the main axis of the cloaca,
which sinus has been interpreted as a repetition of the dorsal
bladder of reptiles. This section also deals with some interesting
anomalies growing out of the attachment of the wolffian ducts to
the cloaca.

A third feature of interest is the regular occurrence in chick
embryos of an accessory bursal diverticulum (div. a), probably
arising from the irregularities consequent upon the formation of
the cloacal fenestra. By means of this diverticulum it has been
possible to define the primordium of the bursa more accurately
than has hitherto been done and therefore to offer new suggestions
regarding its phylogenetic origin.

LITERATURE CITED

BornuaAvurpt, THropor 1867 Untersuchungen iiber die Entwickelung des
Urogenitalsystems beim Hiihnchen. Riga.

Boypren, Epwarp A. 1918 Vestigial gill-filaments in chick embryos with a note
on similar structures in reptiles. Am. Jour. Anat., vol. 23, pp. 205-236.

De Graar, R. 1668 De mulierum organis generationi inservientibus. Lugd.
Batav., vol. 8, p.317. See Taf. 17, Fig. K.

THE CLOACA IN BIRDS 193

Faprictus, HIERONYMUS AB AQUAPENDENTE 1687 Opera omnia anatomica et
physiologica. Lips. De formatione ovi et pulli. p.3.

Feitrx 1906 Die Entwickelung des Harnapparates in Hertwig’s Handb. der
vergl. u. exp. Entw., Bd. 3, Tl. 1, 8S. 483.

FLEISCHMANN, ALBERT 1902 Kloake und Phallus der Amnioten. Morphol.
Jahrb., Bd. 30, S. 539-675.

Forses, W. A. 1877 On the bursa of Fabricius in birds. Proc. Zool. Soc.
London, pp. 304-318.

Gapow, Hans 1889 Cloake und Begattungsorgane. S. 845 in Bronn’s Thier-
reich, Bd. 6, Abt. 4, Aves.

Gasser 1880 Die Entstehung der Kloakenéffnung bei Hiihnerembryonen.
Arch. f. Anat. u. Entwick., S. 297-319.

Harvey, Wintt1AM 1651 De generatione animalium. Tr. Syd. Soc. 1847, p. 183.
London.

Jounson, F. P. 1914 The development of the rectum in the human embryo.
Am. Jour. Anat., vol. 16, pp. 1-57.

Jotty, T. 1915 La bourse de Fabricius et les organes lymphoépithéliaux.
Arch. d’anat. micr. T. 16, pp. 363-547.

KEIBEL, Franz 1902 Zur Anatomie des Urogenitalkanals der Echidna aculeata
var. typica. Anat. Anz., Bd. 22, S. 301.
1921 Der Schwanzdarm und die Bursa Fabricii bei Vogelembryonen.
Anat. Anz., Bd. 54, S. 301-303.

LEwIs AND Parez 1914 Anat. Rec., vol. 9, p. 105.

Leypia 1857 Lehrbuch der Histologie, S. 321. Frankfurt am Main.

Minot, C.8. 1900 On the solid stage of the large intestine in the chick. Bos.
Soc. Nat. Hist., vol. 4, pp. 153-164.
Osawa, GAKuTARO 1911 Ueber die Bursa Fabricii der Végel. Mitt. aus den
Med. Fak. der Kais. Jap. Univ. zu Tokyo, Bd. 9, 8S. 299-341.
PomAYER, Cart 1902 III. Die Vogel. In Fleischmann’s Kloake und Phallus,
S. 78-116.

RertTerer, E. 1885 Contributions a l’etude du cloaque et de la bourse de
Fabricius chez des oiseaux. Journ. de l’anat. et la phys., T. 21, p. 369.
1913 a Nouvelles recherches sur la bourse de Fabricius. C. R. de la
Soc. de Biol., 25 janvier, T. 74, p. 182.
1913 b Homologies de la bourse de Fabricius. C. R. de la Soc.de
Biol., 22 février, T. 74, no. 8, p. 382.

ScHrEINER, K.E. 1902 Ueber die Entwickelung der Amniotenniere. Zeitschr.
f. wiss. Zool., Bd. 71, S. 66.

ScHUMACHER, SIEGMUND 1903 Ueber die Entwicklung und den Bau der Bursa
Fabricii. Sitzber. d. kais. Akad. d. Wiss. in Wien, Bd. 112, Abt. III,
S. 163.

Strepa, Lupwiag 1880 Ueber den Bau und die Entwickelung der Bursa Fabricii.
Zeitschr. f. wiss. Zool., Bd. 34, S. 296-309.

UnrTERHOSSEL, Paun 1902 I. Die Eidechsen und Schlangen, 8. 545. In
Fieischmann’s Kloake und Phallus.

Wencxesacu, K. F. 1888 De Ontwikkeling en de Bouw der Bursa Fabricil.
Proefschrift. Leiden.
1896 Die Follikel der Bursa Fabricii. Anat. Anz., Bd. 11, 8. 159.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 2

PLATE 1
EXPLANATION OF FIGURES

Models illustrating the formation of a cloacal fenestra and the early develop-
ment of the cloaca in chick embryos. All figures are drawn to the same scale
(magnification, X 50). H. F. Aitken, del. (plates 1 and 4).

13 H. E. C. 2071: 2 days, 18 hours (ef. with text fig. 5, a sagittal reconstruc-
tion of the same embryo). In passing across the picture from left to right at its
upper level the organs are encountered in the following order: medullary tube,
notochord, caudal intestine, primitive-streak mass, proctodaeum, allantois,
rectum, dorsal aortae. This stage shows the persistence of a mass of primitive-
streak tissue in the angle between the cloaca and caudal intestine; the mergence
of four structures (medullary tube, notochord, caudal intestine, and anterior
half of primitive-streak remnant) with the indifferent tail-bud mass; a cireclet of
five or six complemental diverticula around the unattached terminal portion of
the W. D.; a larger complemental diverticulum opposite its shaft; and the isolated
foramina (in the back wall of the cloaca and adjacent portion of the caudal intes-
tine) which mark the first step in the disintegration of the cloacal wall and the
formation of a fenestra.

14 and 15 H.E.C. 1958: 3 days, 6 hours; 8 mm. (ef. with fig. 22, a sagittal
reconstruction of the same embryo). At the left of figure 13 are the remnants
of the caudal intestine and primitive streak, each detached from the cloaca by a
process of disintegration. The dash line indicates that portion of the cavity of
the cloaca which has been denuded of epithelium. It is bounded by mesenchyma
only, and indicates the maximum extent of the cloacal fenestra, shown to better
advantage from below in figure 15. At the cephalic end of the fenestra in both
figures is an aberrant diverticulum probably derived from one of the comple-
mental diverticula shown in figure 12.

16 H.E.C.1942:4 days, 3 hours; 10.5mm. (ef. with fig. 27, a sagittal recon-
struction of the same embryo). Note diverticula lettered a and ¢ in fig. 26,
together with accompanying legend. :

17 H.E.C. 2097: 4 days, 3 hours; 10.5 mm. (cf. with fig. 28, a sagittal recon-
struction of the same embryo). Note accessory diverticulum lettered b in figure
27.

18 H.E.C. 1951: 5 days, 13 mm. (ef. with fig. 31, a sagittal reconstruction of
the same embryo). Note the swelling (bursa of Fabricius) caused by coalescence
of vacuoles at the bottom of the cloaca; the distended cavity of the urodaeum
connecting allantois and excretory ducts; the flattened and occluded area between
the urodaeum and bursa (urodaeal membrane); the down-growing proctodaeum
astride the cloacal membrane, reaching out to connect with the bursa; the con-
striction in the metanephric pelvis marking the future division between the
second and third lobes of the adult kidney (ef. with fig. 39).

194

THE CLOACA IN BIRDS PLATE 1

EDWARD A. BOYDEN

y
[

r

———

=.
‘
slieess :

—. ————~

PLATE 2
EXPLANATION OF FIGURES

Projection-lantern drawings of microscopic sections through the cloacal
fenestra of chick embryos, drawn to the same scale (magnification, X 340).

19 H.E.C. 512 (section 121): 2 days, 20 hours? Obliquely-transverse section
passing through cloaca at right angles to the long axis of the fenestra (ef. with
imaginary line connecting letters y and all. in text fig.5). Note bilaterally sym-
metrical gaps in cloacal wall; the concentration of mesenchyma around the gaps;
the isolated floor of the cloaca, with necrotic cells on margin; the phagocytes in
the cavity and the pyenotie nuclei in the epithelium bordering the gap.

20 H.E.C. 2057 (section 736): 3 days, 4 hours; 6.8 mm. Section through
fenestra during period of maximum extent (cf. embryos shown in figs. 14, 15
and 22). Note complete resorption of disintegrating epithelium shown in pre-
ceding figure, the rounded epithelial margins which fail to regenerate, the flat-
tening out of the mesenchyma bordering exposed cavity.

21 H.E.C. 2124 (section 749): 3 days, 18 hours; 8.5 mm. Last stage before
closure showing section through fenestra reduced to small slit (same age as
embryos shown in figs. 23 and 24). Note approximation of two side walls, the
complete absence of regeneration along epithelial margins.

196

THEZCLOACA IN BIRDS PLATE 2
EDWARD A. BOYDEN

197

PLATE 3

EXPLANATION OF FIGURES

Graphic reconstructions of the cloaca of bird embryos drawn to the same scale
(magnification, X 35). Dash lines indicate cavity; dotted lines, vacuoles. This
plate represents chiefly a quantitative study of chick embryos made to demon-
strate the origin of the bursa of Fabricius together with the identity and sig-
nificance of a series of diverticula occurring on the back wall of the cloaca between
the anal plate and the rectum. Diverticulum a represents an accessory diver-
ticulum, arising from the caudal angle of the cloacal fenestra, which regularly
becomes appended to the bursa of Fabricius; b represents an accessory diver-
ticulum, only occasionally present, which arises from the cephalic angle of the
cloacal fenestra and which becomes associated with the urodaeal sinus; ¢ repre-
sents a diverticulum which regularly forms the medial component of the uro-
daeal sinus.

22 Chick embryo, H.E.C.1953. 3 days, 6 hours, 8.0 mm.

23 Chick embryo, H.E.C. 2120. 3 days, 18 hours, 9.2 mm.

24 Chick embryo, H.E.C. 2126. 3 days, 18 hours, 9.5 mm.

25 Chick embryo, H.E.C. 2058. 4 days, 4 hours, 11.0 mm.

26 Chick embryo, H.E.C. 2098. 4 days, 3 hours, 10.0 mm.

27 Chick embryo, H.E.C. 1942. 4 days, 3 hours, 10.5 mm.

28 Chick embryo, H.E.C. 2097. 4 days, 3 hours, 10.5 mm.

29 Chick embryo, H.B.C. 2100. 4 days, 22 hours, 13.0 mm.

30 Chick embryo, H.E.C. 1943. 4 days, 3 hours, 12.0 mm.

31 Chick embryo, H.E.C. 1951. 5 days, 0 hours, 13.0 mm.

32 Chick embryo, H.E.C. 2059. 4 days, 23 hours, 14.0 mm.

33 Chick embryo, H.E.C. 2074. 5 days, 23 hours, 15.0 mm.

34 Chick embryo, H.E.C. 2076. 6 days, 7 hours, 17.3 mm.

35 Chick embryo, H.E.C. 1962. 8 days, 1 hours, 21.5 mm.

386 Sterna hirundo (common tern) H.E.C. 2169. 8.0 mm.

37 Sterna hirundo (common tern) H.E.C. 2115. 10.4 mm.

38 Sterna hirundo (common tern) H.E.C. 2173. 13.4 mm.

ABBREVIATIONS
all., allantois Mull., Miillerian duct
an., anus pelv., pelvis of kidney
an. pl., cloacal membrane (anal plate) —_phal., phallus
bursa, bursa cloacae (of Fabricius) proct., proctodaeum
cauda, inner curvature of tail ps. v., ventral half of primitive streak
cau. A., caudal artery rect., ampulla recti (coprodaeum)
c.7., caudal intestine umb. A., umbilical artery
copr., coprodaeum (ampulla recti) ur., ureter
d., accessory rectal diverticulum urod., urodaeum
div., complementary diverticula ur. m., urodaeal membrane
fen., cloacal fenestra W. D., Wolffian duct

198

THE CLOACA IN BIRDS PLATE 3
EDWARD A. BOYDEN

PLATE 4

EXPLANATION OF FIGURES

39 Model of chick embryo, H.E.C. 1945: 5 days, 15 hours; 15 mm. X 37.
Showing especially the bladder-like enlargement of the allantois in the intra-
embryonic body cavity, the lateral invaginations of the proctodaeum to meet the
bursa of Fabricius (proct.), and the constriction of the metanephric pelvis into
two parts by the umbilical artery.

40 Model of chick embryo, H.E.C. 1962: 8 days, 1 hour; 21.5 mm. X 87.
Note the occluded rectum, the prominent urodaeal sinus (wrod.), and the
elongating bursa.

41 Model of chick embryo, H.E.C. 1967:11 days;31mm. X21. Note differ-
entiation of bursa into stalk and plicated gland, also division of cloaca into the
three transverse parts characteristic of the adult: proctodaeum (ectodermal
origin); urodacum, cloaca proper, receiving urogenital ducts; and the copro-
daeum, rectal ampulla, with its ‘zona columnaris.’

200

THE CLOACA IN BIRDS PLATE 4
EDWARD A. BOYDEN

Resumen por el autor, Ivan E. Wallin.
Sobre la naturaleza de las mitocondrias.

I. Observaciones sobre los métodos de tefido de las mitocondrias
aplicados a las bacterias.

E] autor ha tefido bacterias mediante los métodos mas emplea-
dos para el tenido de las mitocondrias, especialmente el verde
janus. ‘Todos los métodos procados tinen las bacterias. Los
métodos para el tefiido de las mitocondrias, incluso el de colora-
cién vital mediante el verde janus, no son especificos para las
mitocondrias sino que tinen también bien las bacterias.

II. Reacciones de las bacterias a los tratamientos quimicos.

El objeto de estos experimentos ha sido buscar una diferencia
fundamental en el comportamiento de las bacterias y las mito-
condrias bajo la accién de ciertos agentes quimicos empleados para
determinar la naturaleza quimica de las mitocondrias. El autor
no ha encontrado diferencia , fundamental alguna en _ estas
reacciones.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 27

ON THE NATURE OF MITOCHONDRIA

I, OBSERVATIONS ON MITOCHONDRIA STAINING METHODS
APPLIED TO BACTERIA

II. REACTIONS OF BACTERIA TO CHEMICAL TREATMENT

IVAN E. WALLIN

Department of Anatomy and the Henry S. Denison Research Laboratories, University
of Colorado, Boulder

ONE PLATE (NINE FIGURES)

INTRODUCTION

The publication of Altmann’s ‘Bioblast theory’ (’90) stimulated
a new interest in the investigation of cytoplasm. The minute
bodies observed by Altmann in the cytoplasm were thought by
him to be the ultimate units of life, and the cytoplasm itself was
considered a more or less passive and lifeless substance. This
conception of cytoplasm and the contained bodies or granules
has received no support from recent investigators. The bodies
in question have come to be considered normal cytoplasmic
organs by most investigators. They have been described by a
great number of authors under various names. More recently
the term ‘mitochondria,’ first used by Benda (’98), has come into
general usage.

Following the pioneer work of Flemming (’82), Altmann (’90),
and Benda (’98), a massive literature on mitochondria has ac-
cumulated. This literature has dealt chiefly with the presence
or absence of mitochondria in the various types of cells in both
plants and animals. Cowdry (’18) has given an exhaustive re-
view of mitochondrial literature and has summed up the total of
our knowledge of mitochondria.

It is quite apparent, from a perusal of Cowdry’s excellent
review, that we have an exceedingly limited knowledge concern-

203

204. IVAN E. WALLIN

ing the fundamental properties of mitochondria. Attempts have
been made to investigate their physiological properties, but
aside from a possible relationship to chloroplast formation in
plants, nothing definite, apparently, has been established con-
cerning their function. Regarding the chemistry of mitochondria
investigators generally agree that they are of the nature of phos-
pholipins and lipoids and perhaps contain some albumin. The
theory of their chemical nature is based on their reactions to
staining methods and various chemicals. ‘Artificial mitochon-
dria’ were produced by Léwschen (713) by the use of lecithin in
different salt and albumin solutions.

Considerable study has been given to the morphology of
mitochondria. The result of this type of work has led to the
conclusion by Cowdry (18) and others that the form of mito-
chondria is variable and after all of little importance. Two
forms of mitochondria predominate, namely, rod-shaped and
globular forms. Besides these two predominating types various
irregular forms may be found.

An important consideration in the demonstration of mito-
chondria is the technique. This technique warns to the exclusion,
in the chemicals employed, of various solvents of mitochondria,
chief of which are ether, alcohol, and acetic acid. It has not
been claimed for the majority of mitochondria staining methods
that they are specific for mitochondria. This ‘specificity,’ ap-
parently, has reference to other materials in the cell. However,
it must be assumed that these methods, if they are to be of value,
must have a relative specificity for mitochondria. The janus
green vital staining method has been definitely placed in a class
of specific stains for mitochondria by Cowdry (18, p. 48).

The striking resemblance of mitochondria to bacteria is ap-
parent to all who are familiar with the two groups of structures.
This resemblance has been noted by various authors and has
led Cowdry to suggest a division of mitochondrial literature into
two periods: an older literature in which mitochondria were
observed in cells and mistaken for bacteria and a newer literature
in which they have been observed and recorded under various
names.

ON THE NATURE OF MITOCHONDRIA 205

The chief methods employed in cytological studies are based on
the reactions of stains and chemicals on protoplasm. In many
cases a differentiation between cells and cell structures is demon-
strated solely by staining reactions. While such methods may
be criticized on account of the absence of a definitely indicated
specificity, their value cannot be denied, especially in cases where
the difference is pronounced. It is fair to demand when struc-
tures bear so close a resemblance to each other as mitochondria
do to bacteria that some method must be employed that will
differentiate between the two if they are to be considered distinct
structures.

Cowdry (18 p. 72) says: ‘‘It occasionally happens that tissues
prepared for mitochondria have been invaded by bacteria, in
which case the bacteria stain just like the mitochondria by the
Benda method, with iron hematoxylin and with fuchsin methyl
green. I have found that large bacilli contain granules which
stain intensely and apparently specifically with janus green.
They resemble in distribution the so-called polar granules.
Smaller forms often stain diffusely.’ It is not clear from this
statement whether Cowdry means to limit this staining reac-
tion of bacteria to those forms that have invaded cells or if he
implies that bacterial smears fixed and stained by mitochondrial
methods will give the same results. In another place, Cowdry
(18, p. 135), referring to mitochondria, says: ‘Fortunately,
they may be easily distinguished from bacteria by their staining
reactions (particularly to janus green), by their occurrence in
almost all cells, by their behavior and by their lack of independent
motility.”

This latter statement would appear to imply that all bacteria
possess independent motility. This would be contrary to estab-
lished fact in bacteriology. Just what ‘behavior’ of bacteria
is specifically characteristic is not indicated by Cowdry.

Concerning the staining reaction of bacteria to Janus green, I
cannot agree with Cowdry that ‘‘mitochondria are easily dis-
tinguished from bacteria’”’ by this staining method.

The practically universal occurrence of mitochondria in plant
and animal cells points to a fundamental property of these struc-

206 IVAN E. WALLIN

tures. Their nature remains as much a puzzle to-day as when
they were first discovered. It is with a desire to point out
certain similarities between mitochondria and bacteria besides
the similarity of form as well as seek a specific differentiation be-
tween the two structures that these studies have been undertaken,

MATERIAL AND METHODS

The materials used in this investigation have included a large
number of strains of bacteria, some from known pure cultures and
others from various mixed infections. The mixed specimens were
obtained from sputum from hospital patients, pus centrifuged
from urine, pus from a carbuncle, cultures made from the in-
testinal contents of rabbits and kittens, cultures made from lymph
nodes, and from various other sources.

The staining methods employed were: Bensley’s acid fuchsin
methyl green method, Schridde’s modification of Altmann’s
method, Benda’s crystal violet method, the copper hematoxylin
method and the vital janus green method.

In the second part of this study a number of strains of bacteria
were subjected to the action of alcohol, ether, chloroform, acetic
acid, formaldehyde, potassium bichromate, osmic acid, and heat.
The object of these experiments was not to determine the exact
nature of the response of the organisms to these chemicals and
heat, but to determine the effect on the staining reaction of the
bacteria after such treatment. In every case controls were
stained with the same stain used on the experimental prepara-
tions.

The janus green used in the vital staming was one of two lots
that were kindly donated to the author by Professors Bensley
and KE, V. Cowdry. This opportunity is taken to express ap-
preciation for this helpful courtesy. Viable cultures of human
and bovine tubercle bacilli were supplied by Dr. Harry Gauss,
of the National Jewish Tuberculosis Sanitarium in Denver.

I am especially indebted to my colleague Dr. Severance Bur-
rage, of the Department of Pathology, for valuable assistance
and suggestions in this work and also for generous use of bac-
terial cultures in his laboratory.

ON THE NATURE OF MITOCHONDRIA 207

I. OBSERVATIONS ON MITOCHONDRIA STAINING METHODS
APPLIED TO BACTERIA

In the following staining methods in which a fixation preceded
the staining, smears were made in the usual way on the slide.
Before the smears had time to dry they were immersed in the
fixatives of the different methods and later treated according to
the procedure for the particular method. In a few instances
bacteria were centrifuged, fixed en masse, embedded, and sec-
tioned.

The procedure in the janus green vital staining followed the
method used by Cowdry (714) for blood cells.

a. Bensley’s acid fuchsin methyl green method

This method was used according to the directions given by
Bensley (11). It was found that the time for both fixation and
staining could be shortened considerably with excellent results,
obviously due to the more rapid penetration in the bacterial
smears. In a number of instances the method was altered with
a modified Flemming’s fixation. This modified fixative consisted
of osmic and chromic acids in the following proportions: 4 cc.
2 per cent aqueous solution of esmic acid and 6 cc. 1 per cent
aqueous solution of chromic acid. This modification appeared
to give a more rapid fixation and also good staining results with
bacterial smears.

Besides a large number of unknown bacteria, the followmg
strains were subjected to this method: human and bovine tubercle
bacilli, Bacillus coli communis, Bacillus bulgaricus, Bacillus meg-
atherium, Bacillus subtilis, Staphylococcus pyogenes aureus,
Staphylococcus albus, and a pneumococcus.

In every case where this method was used the bacteria were
well stained. In the majority of cases they were sharply stained.

b. Schridde’s modification of Altmann’s method

This method was used only to the extent of determining a
positive staining in a few cases. The same difficulty experienced
in demonstrating mitochondria with this method was experienced

208 IVAN E. WALLIN

with bacteria. Bacillus bulgaricus, Bacillus coli, and Staphylo-
coccus pyogenes aureus were definitely stained by this method.

c. Benda’s crystal violet method

A fairly large number of known and unknown bacteria were
subjected to this method. - Bacteria responded to this method
just as mitochondria do. It gave the sharpest differentiation of
bacteria obtained in any case where mitochondrial methods were
used. Compared with Gram’s stain, for example, on sputum
smears, it gave a sharper differentiation. Here, also, it was
found that the time for fixation and mordanting may be reduced
considerably.

d. Copper hematoxylin method

This method was applied to only a few strains of bacteria. In
some cases the staining of the bacteria was quite faint. This was
particularly true after fixation with Zenker and the formalin-
Mueller used with the Altmann-Schridde method. After Bens-
ley’s and the modified Flemming fixations the bacteria were
stained very sharply by the copper hematoxylin method.

e. Janus green vital staining method

This method was used as prescribed by Cowdry (14) in a
1:10,000 dilution in physiological salt solution. The dye was
first tested by applying it to lymphocytes from a lymph node of
the rabbit. It was found to stain the mitochondria of the
lymphocytes as described by Cowdry.

The following results will serve to indicate the staining re-
action on bacteria:

1. Human bacillus tuberculosis, viable strain. The bacilli
stained rather faintly, the granular forms were easily recognized
on account of the more intense staining of the granules. Ob-
served ten hours after the preparation was made, the bacilli ap-
peared to be stained slightly deeper.

2. Bovine bacillus tuberculosis, viable strain. The bacilli
stained perhaps a little fainter than the human strain. Observed

ON THE NATURE OF MITOCHONDRIA 209

three hours after the preparation was made, the bacilli did not
appear to have absorbed any more of the dye.

3. Bacillus subtilis. A few moments after the preparations
were made, deeply stained granules could be observed in the
bacilli, while the cytoplasm of the bacilli was very faintly stained.
In some bacilli the granules were very small, in others they were
quite large. Figures 1 to 3 are camera-lucida drawings of some
bacilli from these preparations after different lengths of time in
staining.

4. Bacillus megatherium. The preparations contained a great
number of spores besides the bacilli. The spores appeared to
be tinted by the dye. The staining reaction of the bacilli varied
in different preparations, apparently depending upon the age of
the culture. In some eases the cytoplasm was distinctly stained,
while in other cases it was not stained, but contained intensely
stained granules. Figures 4 to 6 represent camera-lucida draw-
ings of bacilli from various cultures with different lengths of
staining time. In one preparation the cytoplasm was quite
intensely stained immediately after application of the dye. When
it was examined three hours later, the majority of the bacilli
had swelled to about three times the normal size and contained
very large intensely stained granules. A drawing was not made
of this preparation and I have been unable to get the same re-
sults again.

5. Unknown bacilli and cocci from a mixed culture. Both
the bacilli and cocci were intensely stained immediately after
preparation was made. There were a number of bacilli that
were unstained. Obviously, it could not be determined in the
preparation if they belonged to the same strain that did absorb
the dye. Observed ten hours after the preparations were made,
a number of the bacilli were swollen and contained large intensely
stained granules, other bacilli were unstained.

6. Unknown bacilli and spores, apparently a pure culture,
made from the intestinal contents of a rabbit. The bacilli were
intensely stained immediately after the dye was applied. The
spores appeared to be tinted by the dye.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 2

210 IVAN E. WALLIN

7. Unknown bacilli and spores, apparently a pure culture made
from the intestinal contents of a five-day-old kitten. The bacilli
were moderately stained, no granules apparent. The spores did
not appear to have absorbed any of the dye.

8. Unknown cocci, culture made from a human throat swab.
The cocci were moderately stained, no granules apparent.

9. Unknown bacilli, culture made from a lymph node of a
rabbit. Apparently not a pure culture. Some bacilli stained
faintly, others quite intensely. Some large bacilli that were
faintly stained contained intensely stained granules.

10. Unknown bacilli and spores, culture made from a lymph
node of a rabbit. The bacilli were moderately stained. The
spores were decidedly tinted by the dye.

11. Unknown bacilli and cocci, culture made from a lymph
node of a rabbit. Bacilli and cocci were moderately stained.

12. Unknown cocci, culture made from a lymph node of a
rabbit. Preparation contained cocci of two sizes. Larger cocci
were intensely stained, the smaller forms were moderately stained.
The difference in staining was also demonstrated in the two forms
when they were stained with Loeffler’s methylene blue.

13. Bacillus coli, pure laboratory culture. The bacilli stained
intensely immediately after the dye was applied, a few forms were
only faintly stained. After the stain had acted for five and a half
hours, the majority of the bacilli were swollen and contained a
single large intensely stained granule. Figures 8 to 9 are
camera lucida drawings of the preparation immediately after it
was made and five and a half hours later.

DISCUSSION

The results recorded above demonstrate that the mitochondrial
methods used are not specific for mitochondria, but that they
also stain bacteria. The intensity of the stain varied with the
different strains of bacteria used and apparently there was a
variation in intensity with the different methods on the same
strain of bacteria. Such variations apparently, also occur with
mitochondria. The janus green vital staining method appeared
to be the most delicate of the methods used.

ON THE NATURE OF MITOCHONDRIA Del

The effect of janus green on tubercle bacilli was contrary to
expectation. On account of the fatty envelope of these forms,
it was to be expected that they might stain more intensely than
any other bacteria. This would imply that fats, waxes, and
lipoids should respond in a like manner to a given stain. Such
an inference may not necessarily be true. However, the proof
that mitochondria are of a lipoidal nature is far from conclusive.
While there is nothing specially indicated as to the chemical
nature of the bacteria that were stained by janus green, it would
appear that one is justified in concluding that these bacteria
and mitochondria do have something in their chemical structure
that is common to all.

The different reactions of a strain of bacteria at different
periods in the life of the culture to janus green is suggestive.
It would appear that janus green has possibilities as a deli-
cate indicator of the physiological state of certain strains of
bacteria.

II. REACTIONS OF BACTERIA TO CHEMICAL TREATMENT

The behavior of mitochondria when subjected to various chemi-
cals and heat has been one of the chief methods used in determin-
ing the nature of these bodies. N. H. Cowdry (717) made
a detailed study of the comparison of mitochondria in plant and
animal cells. The behavior of the two groups of mitochondria
under the influence of various chemicals (ether, alcohol, for-
maldehyde and acetic acid) as well as their morphology was the
method employed in this comparative study. Cowdry concludes
that there is no difference between the mitochondria of plants and
animals.

It must be admitted at the outset that in most instances there
is nothing specifically indicated in the reaction of minute micro-
scopic particles to chemicals. With perhaps a few exceptions,
these reactions are only relative. For example, ether acting upon
tubercle bacilli for a limited time will extract a fat (supposedly
forming an envelope for the bacillus) from the organism. From
such a reaction there is nothing indicated as to the particular
kind of fat that has been dissolved. However, inasmuch as these

212 IVAN E. WALLIN

methods have been used not only in comparing the mitochondria
of plants and animals, but also in determining the approximate
chemical nature of mitochondria, it is necessary in this compara-
tive study of bacteria and mitochondria to also determine the
reaction of bacteria to these chemicals. It must also be admitted
that there is no basis for supposing that all strains of bacteria
should respond in the same way to a given chemical. It has
been indicated by Cowdry and others that all mitochondria
do not respond to a given chemical in the same way.

The methods employed in this study of the reactions of bacteria
to chemicals were designed to retain as much as possible of the
materials resulting from the chemical action. Metal rings coated
with paraffin were sealed to microscopic slides, smears of the
bacteria were then made inside of the rings, and after the chemi-
cals were added cover-glasses were sealed over the rings to
prevent evaporation. After a given time the cover-glasses were
removed and the chemical was permitted to evaporate. When
the smears had thoroughly dried and the paraffin around the
smears had been removed with xylol, a thin film of celloidin was
painted over the smear. The smears were then stained, using
the carbol-fuchsin method for tubercle bacilli preparations and
Pappenheim’s pyronin-methyl green and Loeffler’s methylene
blue for the other preparations. With careful handling in the
staining and washing, the celloidin membrane remains intact
on the slide. Control preparations were made in connection with
every chemical preparation.

For determining the action of ether, chloroform, and heat on
bacteria it is obvious that the paraffin rings could not be used.
In these experiments large quantities of bacteria were placed in
vials and the ether and chloroform added. After four hours
the ether and chloroform were permitted to evaporate considera-
bly. The remains in the vials were then withdrawn with a
pipette, placed on slides and permitted to evaporate to dryness.
For the heat determinations the organisms were placed in vials
with normal salt solution. The vials were then kept at a constant
temperature in an incubator. After half an hour portions of
the emulsion were withdrawn with a pipette and permitted to
evaporate on slides.

ON THE NATURE OF MITOCHONDRIA ONS

The experiments recorded below were repeated a number of
times. In some cases the results were not identical in one set
of experiments. These differences in results were only slight and
apparently of no particular consequence to the object of the
experiments.

The main object in all of the experiments that follow was to
determine the staining reaction of bacteria after treatment with
chemicals and heat.

A. Action of alcohol on bacteria

Aleohol of various strengths was permitted to act on five
different strains of bacteria for a period of five hours.

a. After 95 per cent alcohol. 1. Human tubercle bacilli. Stain
the same as control, granules appear more distinct than in
control.

2. Bovine tubercle bacilli. Stain the same as control, some
crescent forms apparently not observed in control.

3. Bacillus megatherium (with spores).. Bacilli stained fainter
than controls, spores tinted.

4. Bacillus subtilis. Stained more intensely than control.

5. Unknown cocci and bacilli from a lymph-node culture, two
strains of cocci, one intensely stained and the other very faintly
in controls. The cocci appear to be destroyed. Two strains of
bacilli (different in length) not observed in controls were intensely
stained.

b. After 50 per cent alcohol. 1. Human tubercle bacilli. Some
bacilli are very faintly stained, others appear to be slightly
swollen.

2. Bovine tubercle bacilli. Some indication of disintegration,
the bacilli intact were decidedly shrunken.

3. Bacillus megatherium. Bacilli could not be demonstrated
by staining. The spores were decidedly swollen and in many
parts of the field they were coalesced (partially dissolved).

4. Bacillus subtilis. Bacilli could not be demonstrated by
staining. Field contained intensely stained granular debris.

5. Unknown cocci and bacilli from lymph-node culture. Field
full of very minute well-stained cocci (granules?), also a few

214. IVAN E. WALLIN

large well stained cocci. Some of the larger cocci coalesced.
Few exceedingly small well-stained bacilli. Large bacilli un-
stained.

c. After 25 per cent alcohol. 1. Human tubercle bacilli. Bacilli
stain very faintly and appear shrunken. Granules in bacilli
not visible.

2. Bovine tubercle bacilli. Great number of bacilli disin-
tegrated.

3. Bacillus megatherium. Bacilli could not be demonstrated
by staining. Some unstained bacillus-like forms partially coa-
lesced. Spores coalesced, very few distinct in outline.

4. Bacillus subtilis. Granular debris, stained.

5. Unknown cocci and bacilli from lymph-node culture. Cocci
could not be demonstrated by staining. Few small bacilli
stained. :

d. After 10 per cent alcohol. 1. Human tubercle bacilli. Bacilli
appear more granular than control. Some disintegration.

2. Bovine tubercle bacilli. Most bacilli are granular, some
crescent-shaped, some swollen, and some disintegrated. Some
bacilli intact have a purple color.

3. Bacillus megatherium. Some unstained swollen bacilli pres-
ent. Spores coalesced.

4, Bacillus subtilis. Field contains granular debris which has
the appearance of minute cocci.

5. Unkriown cocci and bacilli from lymph-node culture. Few
intensely stained cocci, bacilli unstained.

e. After 5 per cent alcohol. 1. Human tubercle bacilli. Only
a few bacilli intact and stained in thick part of smear, rest of
field contains debris of disintegration.

2. Bovine tubercle bacilli. Some disintegration. Appear bet-
ter preserved than after action of 10 per cent alcohol.

3. Bacillus megatherium. Bacilli could not be demonstrated
by staining. Spores completely coalesced.

4. Bacillus subtilis. Only slight indication of a few unstained
bacilli.

5. Unknown cocci and bacilli from lymph-node culture.
Granular debris that appears like minute cocci. Few minute
bacilli stained.

ON THE NATURE OF MITOCHONDRIA 215

f. After 2 per cent alcohol. 1. Human tubercle bacilli. AI-
most completely disintegrated. Few swollen poorly stained
bacilli present in field.

2. Bovine tubercle bacilli. Almost completely disintegrated.
Few swollen poorly stained bacilli present in field.

3. Bacillus megatherium. Bacilli could not be demonstrated
by staming. Spores coalesced.

4, Bacillus subtilis. Few intensely stained fragmented bacilli
present in field.

5. Unknown cocci and bacilli from lymph-node culture. This
preparation appears very much like the control. Bacilli ap-
parently not stained. |

B. Action of chloroform and ether on bacteria

a. After chloroform. 1. Human tubercle bacilli. Bacilli in-
tact, but appear shrunken and more granular than control.

2. Bovine tubercle bacilli. Bacilli more faintly stained and
appear more granular than controls.

3. Unknown bacilli, culture from intestinal contents of five-
day-old kitten, two strains of bacilli, large and small. Large
bacilli more granular than control, smaller forms clear and faintly
stained.

4. Bacillus megatherium and spores. Few poorly stained and
shrunken bacilli present. Spores not visible.

5. Staphylococcus albus. No normal cocci visible. Remains
appear like exceedingly minute cocci.

b. After ether. 1. Human tubercle bacilli. Bacilli could not
be demonstrated by staining. Remains, granular debris.

2. Bovine tubercle bacilli. Bacilli could not be demonstrated
by staining. Remains, granular debris.

3. Unknown bacilli, culture from intestinal contents a five-
day kitten. Bacilli could not be demonstrated by staining.
Granular debris appears like minute cocci. .

4, Bacillus megatherium and spores. Few disintegrated
bacilli, remains mostly granular debris. Spores were not visible.

5. Staphylococcus albus. Remains, granular debris.

216 IVAN E. WALLIN

C. Action of acetic acid on bacteria

Acetic acid of various strengths was permitted to act on bacteria
for a period of six hours. Glacial acetic acid was diluted with
distilled water for the various dilutions.

a. After 0.5 per cent acetic acid. 1. Human tubercle bacilli.
Bacilli disintegrated. Granular remains intensely stained
(black).

2. Bovine tubercle bacilli. Many bacilli retain their form,
others disintegrated. Intensely stained.

3. Unknown bacilli, culture from intestinal contents of kitten.
Bacilli unstained, swollen, and coalesced.

4. Bacillus megatherium. Bacilli could not be demonstrated
by staining. Spores swollen.

5. Staphylococcus albus. Swollen, unstained, and partially
coalesced.

b. After 1 per cent acetic acid. 1. Human tubercle bacilli.
Bacilli could not be demonstrated by staining. Remains granu-
lar, intensely stained.

2. Bovine tubercle bacilli. Bacilli could not be demonstrated
by staining. Remains granular, faintly stained. ro

3. Unknown bacilli, culture from intestinal contents of kitten.
Bacilli unstained, swollen, and distorted.

4, Bacillus megatherium. Bacilli unstained and distorted.
Many forms contain ‘bleb’-like swellings at end or center of
bacillus, some contain two or three outpushings. Figure 7 is
a free-hand drawing of a few of these bacilli.

5. Staphylococcus albus. Appear partially dissolved and coa-
lesced. Unstained.

c. After 3 per cent acetic acid. 1. Human tubercle bacilli.
Form of bacilli partially preserved. Faintly stained.

2. Bovine tubercle bacilli. Bacilli appear quite normal and
well stained. Some appear to have vacuoles.

3. Unknown bacilli, culture from intestinal contents of kitten.
Bacilli could not be demonstrated by staining. Remains, amor-
phous and faintly stained.

4. Bacillus megatherium. Few unstained bacilli that appeared
partially dissolved. Spores coalesced.

Mate

ON THE NATURE OF MITOCHONDRIA 217

5. Staphylococcus albus. Cocci unstained and coalesced.

d. After 5 per cent acetic acid. 1. Human tubercle bacilli.
Bacilli could not be demonstrated by staming. Remains, minute,
intensely stained granules.

2. Bovine tubercle bacilli. (Accidentally destroyed.)

3. Unknown bacilli, culture from intestinal contents of kitten.
Bacilli could not be demonstrated by staining. Remains, amor-
phous and faintly stained.

4. Bacillus megatherium. Bacilli and spores unstained and
coalesced.

5. Staphylococcus albus. Cocci unstained and coalesced.

D. Action of formaldehyde on bacteria

Formaldehyde of various strengths (diluted in distilled water)
was permitted to act on bacteria for a period of six hours.

a. After 1 per cent formaldehyde. 1. Human tubercle bacilli.
Bacilli could not be demonstrated by staining. Remains, in-
tensely stained amorphous masses.

2. Bovine tubercle bacilli. Poorly stained bacilli that appear
shrunken.

3. Unknown bacilli, culture from intestinal contents of kitten.
Bacilli could not be demonstrated by staining.

4, Bacillus megatherium. Few unstained and swollen bacilli.
Spores greatly swollen.

5. Staphylococcus albus. Cocci faintly stained and swollen.

b. After 3 per cent formaldehyde. 1. Human tubercle bacilli.
Bacilli could not be demonstrated by staining. Remains in-
tensely stained amorphous masses. —

2. Bovine tubercle bacilli. Bacilli distorted in various ways:
shrunken, crescent-shaped, and some with large intensely stained
granules.

3. Unknown bacilli, culture from intestinal contents of kitten.
No distinct bacilli stained. Unstained spore-like forms partially
dissolved.

4. Bacillus megatherium. Bacilli could not be demonstrated
by staining. Spores partially dissolved.

218 IVAN E. WALLIN

5. Staphylococcus albus. Cocci unstained and smaller than
control.

c. After 5 per cent formaldehyde. 1. Human tubercle bacilli.
Bacilli could not be demonstrated by staining. Remains, in-
tensely stained, large granules.

2. Bovine tubercle bacilli. Bacilli could not be demonstrated
by staining. Remains, granular.

3. Unknown bacilli, culture from intestinal contents of kitten.
Some large and small bacilli present that stain more intensely
than control, also some swollen and unstained forms.

4, Bacillus megatherium. Bacilli unstained and partially dis-
solved. Spores unstained and coalesced.

5. Staphylococcus albus. Cocci swollen, unstained, and
coalesced.

E, Action of potassium bichromate on bacteria

Various strains of bacteria were subjected to the action of potas-
sium bichromate in various concentrations for a period of four
hours. The potassium bichromate was dissolved in distilled
water.

a. After 0.5 per cent solution of potassium bichromate. 1.
Human tubercle bacilli. Bacilli intact could not be demonstrated
by staining. Remains, intensely stained granules.

2. Bovine tubercle bacilli. A few bacilli still retain form.
Remainder of remains intensely stained granules.

3. Staphylococcus pyogenes aureus. Normal cocci could not
be demonstrated by staining. Remains, intensely stained minute
"eaeer:

4. Bacillus megatherium and spores. Bacilli could not be
demonstrated by staining. Spores swollen and stained.

5. Unknown cocci. Some cocci swollen and stained.

b. After 1 per cent potassium bichromate. 1. Human tubercle
bacilli. Bacilli could not be demonstrated by staining. Granu-
lar remains intensely stained.

2. Bovine tubercle bacilli. Bacilli could not be demonstrated
by staining. Granular remains intensely stained.

ON THE NATURE OF MITOCHONDRIA 219

3. Staphylococcus pyogenes aureus. Cocci swollen and par-
tially destroyed, also stained.

4, Bacillus megatherium and spores. Bacilli could not be
demonstrated by staining. Spores unstained, some swollen.

4. Bacillus megatherium and spores. Bacilli and spores
preserved, but unstained.

5. Unknown cocci. Few swollen and stained cocci. Clumps
of stained granular debris.

c. After 2.5 per cent potassium bichromate. 1. Human tubercle
bacilli. Bacilli intact could not be demonstrated by staining.
Granular remains minute particles and intensely stained.

2. Bovine tubercle bacilli. Few faintly stained bacilli intact.

3. Staphylococcus pyogenes aureus. Cocci could not be dem-
onstrated by staining. Granular remains.

4, Bacillus megatherium and spores. Few swollen and un-
stained bacilli. Outline of spores very faint.

5. Unknown cocci. Cocci very faintly stained and swollen.

F., Action of osmic acid on bacteria

Various strains of bacteria were subjected to the action of 1
per cent and 2 per cent osmic acid for a period of four hours.

a. After 1 per cent osmic acid. 1. Human tubercle bacilli.
Bacilli well preserved, stain purple.

2. Bovine tubercle bacilli. Bacilli well preserved, stain deep
red.

3. Staphylococcus pyogenes aureus. Cocci preserved, but
unstained.

4, Bacillus megatherium and spores. Bacilli and spores pre-
served, but unstained.

5. Unknown cocci. Could not be seen on the slide.

b. After 2 per cent osmic acid. 1. Human tubercle bacilli.
Poorer preservation than with 1 per cent osmic, faintly stained.

2. Bovine tubercle bacilli. Well preserved and intensely
stained.

3. Staphylococcus pyogenes aureus. Cocci could not be seen
on the slide. |

220 IVAN E. WALLIN

4, Bacillus megatherium and spores. Bacilli and spores un-
stained, outlines difficult to see. .
5. Unknown cocci. Preserved, but unstained.

G. Action of moist heat on bacteria

Various strains of bacteria were placed in vials containing
physiological salt solution and kept in an oven at a constant
temperature of 49°C.

a. After thirty minutes at 49°C. 1. Human tubercle bacilli.
Bacilli could not be demonstrated by staining. Field apparently
contained fat globules.

2. Bovine tubercle bacilli. Bacilli could not be demonstrated
by staining. Field contained granular remains, stained amor-
phous masses and apparently fat globules.

3. Staphylococcus pyogenes aureus. Cocci could not be dem-
onstrated by staining. Remains very minute granules.

4, Bacillus megatherium and spores. Bacilli could not be
demonstrated by staining. Spores coalesced.

5. Unknown cocci. Cocci could not be demonstrated by stain-
ing. Remains, stained amorphous masses.

DISCUSSION

The results obtained from these experiments demonstrate that
bacteria may lose their staining properties when subjected to the
action of certain chemicals ordinarily used in microscopical tech-
nique. The degree to which the staining reactions were affécted
varied with the different chemicals and also with the strain of
bacteria.

In many cases the bacteria retained their form, but were
unstained, and in other experiments the bacteria were fragmented.
In the cases where the organisms could not be seen they ap-
parently had been dissolved or fragmented. In the majority of
experiments where the remains on the slide were granular and
fragmented these remains were stained. The possibility suggests
itself that mitochondria may behave in the same way and that
some of the irregularly shaped mitochondria sometimes observed
may be the fragments resulting from chemical action.

ON THE NATURE OF MITOCHONDRIA 220

The visibility of unstained bacteria varies with the difference
in refraction of the bacteria and the surrounding medium. In
those cases where the bacteria could not be seen the granular
remains indicated the destruction of the organism. Unstained
bacteria lodged in the cytoplasm of tissue cells cannot be dis-
tinguished easily and in some cases they are not visible. It is
generally supposed that mitochondria are dissolved by the action
of certain chemicals. It is possible that in many cases where
they cannot be demonstrated by staining their form has been
retained, but unstained and consequently not readily observed.

Bacteria apparently respond to heat in the same way that
mitochondria do. The end-product from the action of heat was
not the same for all the strains of bacteria that were used for this
experiment. In some cases the remains were granular, in others
they were amorphous. The amorphous material apparently
represented the residuum of a solution after evaporation.

Cowdry (18, p. 68) has noted the presence in some secreting
cells of mitochondria with ‘bleb-like’ swellings and in egg cells
of ‘dumb-bell-shaped’ mitochondria. The action of 1 per cent
acetic acid on Bacillus megatherium is significant in this connec-
tion. The imitation of such ‘bleb-like’ and ‘dumb-bell-shaped’
mitochondria by bacteria as the result of chemical action sug-
gests the possibility that mitochondria of these types may be
due to the action of the chemicals used in fixation.

CONCLUSIONS

The results obtained in subjecting bacteria to mitochondrial
staining methods and to the chemicals that have been utilized
to determine the chemical nature of mitochondria appear to
demonstrate that these methods are not specific for mitochondria,
but have a similar reaction on bacteria. To the degree that these
staining methods and chemical reactions are not specific, bacteria
and mitochondria have a similar chemical constitution.

Dip Ne IVAN E. WALLIN

LITERATURE CITED

ALTMANN, R. 1890 Die Elementarorganismen und ihre Beziehungen zu den
Zellen. Leipzig.

Brenpa, C. 1898 Ueber die Spermatogenese der Vertebraten und héheren Inver-
tebraten. 2. Die Histogenese der Spermien. Verh. d. physiol. Ges.

Brnsuey, R. R. 1911 Studies on the pancreas of the guinea-pig. Am. Jour.
Anat., vol. 12.

Cowpry, E. V. 1914 The vital staining of mitochondria with janus green and
diethylsafranin in human blood cells. Intern. Monatschrift f. Anat.
u. Phys., Bd. 31.
1918 The mitochondrial constituents of protoplasm. Carnegie Inst.
Publications, Contrib. to Embryology, vol. 8, no. 25.

Cownpry, N. H. 1917 A comparison of mitochondria in plant and animal cells.
Biol. Bull., vol. 33.

FLEMMING, W. 1882 Zellsubstanz, Kern und Zellteilung. Leipzig.

Loéwscuin, A. M. 1913 Myelinformen und Chondriosomen. Ber. d. deutsch.
bot. Ges., Bd. 31.

PLATE
EXPLANATION OF FIGURES

All the figures, with the exception of figure 7, were made with the aid of the
camera lucida. They were all drawn to the same scale. The lenses used were:
2-mm aproch. oil-immer. obj., comp. ocular no. 8.

1 Bacillus subtilis from an old culture one hour after the application of Janus
green.

2 Bacillus subtilis, a) 5} hours after application of janus green; b) 20 hours
after application of janus green.

3 Bacillus subtilis from a 48-hour culture 24 hours after application of janus
green.

4 Bacillus megatherium from an old culture 5} hours after application of
janus green. ‘

5 Bacillus megatherium from a 20-hour culture 15 minutes after application
of janus green.

6 Bacillus megatherium from an old culture } hour after application of janus
green.

7 Free-hand drawing of bacillus megatherium after action of 1 per cent acetic
acid for a period of six hours.

8 Bacillus coli 15 minutes after application of Janus green.

9 Bacillus coli (same specimen as in fig. 8) 5} hours after application of janus
green.

ON THE NATURE OF MITOCHONDRIA PLATE 1
IVAN E. WALLIN

ON THE NATURE OF MITOCHONDRIA 225

ADDENDUM

After this paper was submitted for publication, my attention was
called to a work by Portier (18) entitled, ‘‘Les Symbiotes,” and to
criticisms of Portier’s book by Regaud (719) and Guilliermond (’19).

Unfortunately, I have not been able to secure a copy of Portier’s
book in time to review it in this article. However, I have perused
Regaud’s and Guilliermond’s criticisms. From these criticisms it is
apparent that Portier in 1918 stated a theory regarding mitochondria
that coincides with a conception of these bodies that has been growing
in my own mind. I was not ready to state this hypothesis until I
had collected more evidence in its support. <A brief consideration of
Regaud’s and Guilliermond’s criticisms is pertinent at this time.

Obviously, the details of Portier’s evidence cannot be considered in

this discussion. Regaud quotes the following resumé from Portier’s
“Les Symbiotes”: ‘‘Chaque cellule vivante renferme dans son proto-
plasme des formations que les histologists désignent sous le nom de
mitochondries. Ces organites‘ne seraient pour mio autre chose que des
bactéries symbiotiques, ce que je nomme des symbiots. .
La bactérie symbiotique vient du milieu extérieur: elle peut, dans
certains cas, y retourner et vivre d’une vie indépendante. Les bac-
téries seraient done les seuls étres simples; tous les autres seraient
doubles.”

Regaud indicates various characteristics of mitochondria that are
supposedly not shared by bacteria. He mentions the inconstancy of
form of mitochondria, their behavior towards acids and metallic salts,
their albumin-lipoid constitution, their fragility, the impossibility of
mechanical extraction of mitochondria from the living cell, and the
synthetic properties of mitochondria. He also indicates the following
characteristics of bacteria that presumably, are not shared by mito-
chondria: bacteria are definite organisms having a stable form (diffi-
cult to change in shape); bacterial life is generally resistant to chemical
and physical agents; bacteria are easily extracted mechanically from
living cells without alteration of the form of the bacteria; the form and
structure of bacteria and even their staining qualities are indifferent to
fixation.

I shall discuss briefly each of these characters that Regaud considers
distinctive.

1. Inconstanecy of form of mitochondria. This cannot seriously
be considered a characteristic of mitochondria. It is a well established
fact in bacteriological technique that certain bacteria assume different
forms in different media (Jordan, ’20, p. 67).

2. The behavior of mitochondria towards acids and metallic salts.
In the second section of this paper I have given sufficient evidence that

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 2

226 IVAN E. WALLIN

some bacteria behave like mitochondria to acetic acid and other chemi-
‘als. The behavior of bacteria after fixation with a fixative containing
potassium bichromate is definitely indicated by their staining reaction
and is not unlike mitochondria.

3. The albumin-lipoid constitution of mitochondria. The chemical
nature of mitochondria is unknown. I have shown in the second seec-
tion of this paper that the chemical reactions used in attempting to de-
termine the chemical nature of mitochondria have a similar effect
on some bacteria.

4. The fragility of mitochondria. Mitochondria vary in fragility.
This, I believe, has been assumed by various investigators. In a paper
in preparation I will definitely demonstrate this difference. This
character of mitochondria, per se, is in favor of a bacterial nature of
mitochondria. Biological data furnish many examples of plants and
animals that have become ’fragile’ as a result of well-developed sym-
biosis and parasitism. The tapeworm is an example of a comparatively
fragile organism. Its relationship to host is not as intimate as an in-
tracellular symbiotic bactertum would be to its host. Surrounded by
a living cytoplasmic environment, it is to be expected that a well-
established symbiotic organism would lose many properties that the
genetic type possessed. Further, one would expect the symbiotic form
to acquire new properties.

5. The impossibility of mechanical extraction of mitochondria
from the living cell. This apparent difficulty is undoubtedly dependent
upon the fragility of mitochondria and consequently is irrelevant to
the real problem.

6. The synthetic nature of mitochondria. Regaud argues that
mitochondria are unlike bacteria in that they exhibit synthetic prorer-
ties in the cytoplasm. He quotes himself and other investigators
to support the contention that mitochondria produce secretion granules,
pigment, etc. It appears to me that Regaud’s argument is at least,
equally convincing evidence that mitochondria are organisms.

7. Bacteria are definite organisms having a stable form. Bacterio-
logical evidence does not support this contention. “The tubercle
bacillus, for example, under ordinary conditions, is a typical rod, but
sometimes produces branching filaments, and has been placed by some
writers with the trichomycetes” (Jordan, ’20, p. 64).

8. Bacterial life is generally resistant to chemical and physical agents.
The chemical and heat experiments recorded in the second section of
this paper refute this statement. Bacteria are not only affected by
these agents, but in some cases are extremely sensitive to them.

9. Bacteria are easily extracted mechanically from living cells with-
out alteration of the form of the bacterium. This argument has no
bearing on the problem for, a priori, it must be admitted that a bacte-
rium that develops an intracellular symbiotic existence would acquire

fragility.

ON THE NATURE OF MITOCHONDRIA Dat

10. The form and structure of bacteria and even their staining
qualities are indifferent to fixation. This is not in agreement with the
results recorded in the second section of this paper. It was found that
not only the form was altered by the fixation, but the staining qualities
were also distinctly altered. In the paper in preparation I shall give
further evidence regarding this point.

Regaud states that absolute differences between bacteria and mito-
chondria are incontestable. Further, he demands that if Portier
will not admit that there are differences, he will have to demonstrate
that these two structures (mitochondria and bacteria) can change
from one to the other. I cannot find in Regaud’s criticism the ‘ab-
solute differences’ which he claims are incontestable. Regarding the
demand for a demonstration of reversibility of mitochondria and bac-
teria as a proof that mitochondria are organized entities, it seems to me
that one has just as much ground to demand that it be demonstrated
that a tapeworm can revert to a free-living organism to establish its
individuality.

Portier (19) answers the arguments advanced by Regaud. Not
having a full knowledge of Portier’s data, I shall not attempt to con-
sider Portier’s rebuttal. However, one argument advanced by Portier
in explaining the source of his ‘symbiotes’ (mitochondria) invites a
critical consideration. Portier found bacteria in the intestine of the
rabbit and apparently found similar bacteria in the cytoplasm of the
intestinal epithelial cells. From this observation Portier concludes that
the source of ‘symbiotes’ is from the intestinal contents. Regaud,
justly, refuses to accept this interpretation of the phenomenon. I
have observed the same phenomenon in the intestinal contents and the
intestinal epithelial cells in a one-day-old kitten after mitochondrial
fixation and staining. The fact that mitochondria may be demonstrated
in the cells of embryos before the intestine 1s formed excludes the pos-
sibility of such an origin. The question of the origin of mitochondria
is a major problem that may well rest until the nature of mitochondria
has been extablished.

Guilliermond (719) discusses three sections of Portier’s book. I
shall briefly discuss his criticisms in the order given.

1. Analogous forms. Guilliermond admits the analogy of form and
further admits that mitochondria exhibit the property of division. He
calls attention to the fact that the slightest upset in osmotic equilibrium
suffices to change the character of mitochondria. In hypotonic media
mitochondria immediately swell and transform into large vesicles.
Of most important value as evidence, he argues that mitochondria have
very little resistance to alcohol, chloroform, and acetic acid, and that
it has been shown that a temperature of 40°C.! is sufficient to destroy
the mitochondria in a few moments. He also says: “Up to the present
time bacteria are not known that exhibit such fragility.”

1 In a correction (Compt. rend. des Soc. Biol., T. 82, p. 396), Guilliermond
changes the temperature at which mitochondria disappear to 47°C.

228 IVAN E. WALLIN

This criticism deals with the fragility of mitochondria. I have
answered this criticism above. However, I again insist that even
if such a difference were a reality, it, has no bearing on the problem.
It must be admitted, on the basis of known biological behavior,
that fragility is an accompaniment to well-established symbiosis and
parasitism.

2. Bacteria are stained like mitochondria by Regaud’s method.
Portier’s ‘symbiotes’ resist alcohol and acetic acid, are stained easily
without change after fixation. Guilliermond states that this fact is an
excellent means of differentiating between mitochondria and ‘symbiotes.’
Guilliermond admits that some mitochondria are more resistant than
others to acetic acid and alcohol, but maintains that the more resistant
forms are no longer mitochondria, but plastids differentiated from
mitochondria.

In the first section of this paper I have shown that bacteria are stained
like mitochondria by a number of mitochondrial methods, including the
vital janus green method. From Guilhermond’s criticism it would
appear that Portier’s ‘symbiotes’ are not mitochondria, but some other
organism. In my work on staining of bacteria with mitochondrial
methods I have found no fundamental difference in the staining reac-
tions of mitochondria and bacteria. Guiliermond’s statement that the
more resistant mitochondria are no longer mitochondria needs elucidat-
ing evidence. If mitochondria metamorphose into organs that exhibit
synthetic properties, then they assume properties that are characteris-
tic of organisms. Numerous investigators have observed that mito-
chondria differ in their power of resistance to acetic acid and alcohol.
I have shown in the second section of this paper that bacteria also
differ in their reactions to these chemicals.

3. Mitochondria may be cultivated in certain cases. Guiliermond
does not accept Portier’s statement that he has grown mitochondria.
He concludes with the statement: “We cannot conceive that anyone
can culture such fragile elements.”’

I am not in a position to intelligently consider this latter criticism
at this time. On the basis of evidence that I shall submit in a paper
in preparation, I feel confident that mitochondria may be transferred
intact to culture media. While it must be admitted that a demonstra-
tion of mitochondria growing as independent organisms in a culture
medium would be absolute proof of their organized or bacterial nature,
the lack of such a demonstration is not proof that they are cytoplasmic
organs and not organisms.

The writer will discuss Regaud’s and Guilliermond’s criticisms at
_~more length in a paper in preparation. From the preceding discussion,
it is apparent that the fundamental aspects of the problem are not
clearly defined. This confusion is apparently due to the vagueness
of mitochondrial and bacterial definitions. Mitochondrial literature
has not supplied a satisfactory definition of mitochondria. Jordan’s
“General Bacteriology”? does not contain a definition of bacteria.

ON THE NATURE OF MITOCHONDRIA 229

LITERATURE CITED

GuILLIERMOND, A. 1919 Mitochondries et symbiotes. Compt. rend. des Soc.
Biol., T. 82, pp. 309-312.

JoRDAN, Epwin O. 1920 General bacteriology. Philadelphia.

Portier, P. 1918 Lessymbiotes. Masson, Paris.
1919 Discussion, Comp. rend. des Soc. Biol., T. 82, pp. 247-250.

Reagaup, C. 1919 Mitochondries et symbiotes. Compt. rend. des Soc. Biol.,
T. 82, pp. 244-251.

Resumen por la autora, Beatrice Whiteside.
El desarrollo del saco endolinfatico de Rana temporaria Linné.

Este trabajo trata del desarrollo del.saco endolinfatico de la
rana desde la época en que aparece esta estructura hasta que
adquiere la forma hallada en el adulto. La autora ha escojido
la rana porque en este animal el saco es mds grande y complicado
de forma que en cualquier otro vertebrado investigado hasta el
presente. La primera parte del trabajo consiste en una descrip-
cidn de los estados sucesivos del desarrollo con especial referencia
a la histologia de la estructura mencionada y a su situacti6n re-
specto a las meninges espinales. Despues pasa a describir la
topografia e histologia del 6rgano después de haber completado
su desarrollo. La segunda parte del trabajo esta dedicada a una
descripcién de la estreutrua anat6émica del saco en todas las clases
de los vertebrados.

Translation by José F. Nonidez
Cornell Medical College, New York

estas

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, FEBRUARY 27

THE DEVELOPMENT OF THE SACCUS ENDOLYM-
PHATICUS IN RANA TEMPORARIA LINNE

BEATRICE WHITESIDE
Zoological Laboratory, University of Zurich
NINETEEN FIGURES

INTRODUCTION

The membranous labyrinth of vertebrates has often been made
the object of close study. There is one structure, however,
connected with the ear which has hitherto received very little
attention, namely, the saccus endolymphaticus. It is true that
the topography of this organ is well known in most animals, but
we are as yet badly informed as to its development, histology,
and function.

Under these circumstances, I have, at Professor Hescheler’s
suggestion, undertaken an investigation of the development of
the saccus endolymphaticus in Rana temporaria Linné. The
work was done in the Zoological Laboratory of the University of °
Zurich, Switzerland, under the guidance of Prof. K. Hescheler,
and I wish to take this opportunity of thanking him for the assis-
tance he has so kindly given me. I also wish to express my
thanks to Prof. J. Strohl, Dr. Marie Daiber, and Dr. H. Steiner,
of Zurich University, for their many helpful suggestions, and
I am indebted to Mr. A. Bychowsky for the execution of figures
LI to:.19.

The present paper is divided into two principal parts: First,
a description of the development of the saccus endolymphaticus
in Rana temporaria Linné, and, second, an account of the topog-
_ raphy of this structure in the different classes of vertebrates
‘and the conclusions to be drawn from the same.

In order to show the relation of the saccus endolymphaticus
to the other parts of the labyrinth, I shall preface my report
with a short summary of Gaupp’s (’04) description of the
frog’s ear.

231

2352.0" BEATRICE WHITESIDE

In the membranous auditory organ of the frog the following
parts can be distinguished: (fig. 1) the utriculus (utr.) with
the sinus superior (st.swp.utr.) and posterior (s?.post.utr.), the
recessus utriculi (rec.utr.), the three semicircular canals (ca.s.c.)
with the ampullae (amp.), the sacculus (sac.) with the ductus
(d.e.) and saccus endolymphaticus (s.e.), the pars neglecta
(p.negl.), the pars basilaris (p.bas.), the lagena cochleae (lag.),
and the tegumentum vasculosum (not visible in the figure.)

The utriculus is irregularly cylindrical in form, with its long
axis running horizontally from back to front. It is situated
close to the median side of the bony labyrinth. On one side the
utriculus passes into the recessus utriculi; on the other, into the
narrow part of the sinus posterior. The three semicircular
canals arise from the utriculus and each one is connected with
this organ by two ends, the crus simplex and the crus ampullare
(amp.), the former not being any wider than the canal itself,
whereas the latter expands into an oval sac. The utriculus
communicates with the sacculus through the foramen utriculo-
sacculare and is connected at the latter’s circumference with the
sacculus and the pars neglecta. According to Gaupp, thesacculus
of the inner ear has the shape of an oval sac, and possesses four
pouch-like enlargements, namely, the lagena, the pars basilaris,
the pars neglecta, and the tegumentum vasculosum. The
auditory nerve enters the labyrinth with its two branches, the
ramus anterior (r.ant.) and the ramus posterior (r.post.). It
ends in the three cristae acusticae of the ampullae and also in
the macula recessus utriculi, the macula sacculi, the macula
lagenae, the macula neglecta, and the papilla basilaris. Each of
the three first-mentioned maculae is covered with a membrane,
on which many lime crystals are deposited. At its upper median
side the wall of the sacculus expands into the ductus endolym-
phaticus (recessus labyrinthi, aequaeductus vestibuli). This
structure is a very long and narrow canal which runs upwards
median to the utriculus, and leaves the bony auditory capsule
through a special aperture, the foramen endolymphaticum (aper-
tura aequaeductus vestibuli). Inside the cranial cavity it
widens (Hasse, ’73) into the large saccus endolymphaticus,

ee

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 233

which surrounds the brain and extends caudally into the verte-
bral canal. The part lying within the vertebral canal sends out
processes through the intervertebral foramina which come to
lie upon the spinal ganglia (Coggi, ’90). All the different parts

& He titay, a
Ww sll Wii. w.€2:SC
ca.Sc G@. AQrest:
ant. .

os Ze

Le, Gy; = SS
Hi].

A i

WN

: hua )

“A
AUTH

*~npost.N.W
“lag.

~~. Sac. |

Fig.1 Membranous labyrinth of Rana, according to Retzius (taken from
Gaupp 96). amp.ant., ampulla anterior; amp.post., ampulla posterior; ca.sc.ant.,
canalis semicircularis anterior; ca.sc.lat., canalis semicircularis lateralis; ca.sc.-
post., canalis semicircularis posterior; d.e., ductus endolymphaticus; lag., lagena;
mac.sac., macula acustica sacculi; p.bas., pars basilaris; p.negl., pars neglecta;
r.ant.N. VIII, ramus acusticus anterior;r. post. N. VIII, ramusacusticus posterior;
rec. utr., recessus utriculi; sac., sacculus; s7. post. utr., sinus posterior utriculi;
st. sup.ulr., sinus superior utriculi; wér., utriculus.

of the saccus endolymphaticus are filled with a milky fluid con-
taining many lime crystals which refract light and, according
to Sterzi (99), exhibit brownian movement.

My first task was the investigation of the development of the
saccus endolymphaticus, especially in regard to its histological

234 BEATRICE WHITESIDE

conditions and the first appearance of the calcareous contents.
My paper begins with the stage at which Krause’s investigation
(01) closes, in which the auditory vesicle is divided into utricu-
lus and sacculus. At this time the ductus endolymphaticus is a
small canal leading up from the sacculus, and the saccus is indi-
cated by a small expansion at the distal end of the ductus.

Before starting a report of my own investigation, I shall give
a short account of previous observations on the development of
the endolymphatic organs in the common frog, beginning with
Krause (’01), who describes the formation of the ductus as follows.
The auditory plate is formed from the inner layer of ectoderm on
either side of the hind-brain. It consists of a single layer of long
cylindrical cells which are longest in the middle of the plate,
decrease in length toward its sides, and eventually pass into the
very low cells of the outer layer of the ectoderm. First the
dorsal side of this plate bends downwards and grows ventrally,
losing its connection with the outer layer of the ectoderm. Thus
the dorsal part of the auditory vesicle becomes marked off. This
first formed part is the rudiment of the ductus endolymphaticus.
Then the ventral:side follows, bulging outward at the same time.
In this way the ductus is still more distinctly separated from the
rest of the auditory vesicle and forced to the latter's median
surface.

There are, to my knowledge, only three other papers on this
subject, namely, those of Villy (90), Poli (97), and Corning
(99). All three investigators agree that the organ is formed in
the above described manner.

In all vertebrates the saccus endolymphaticus originates as
an expansion of the distal part of the ductus. This structure
is therefore a part of the inner ear and has no connection with the
lymphatic spaces lying within the skull. Most authors confine
themselves to this statement, and I know of only two detailed
reports on the further development of this organ, the one by
Rothig and Brugsch (’02), on the chick, the other by Streeter
(16), on the human embryo.

As regards the frog in particular, the papers of Coggi (’90)
and Villy (90) are to be mentioned. Coggi gives a few details

EEF ee

=

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 235

which I shall quote in the course of my paper, while all that
Villy writes on the subject is the following paragraph:

Until the semicircular canals are formed, little is to be noticed regard-
ing the ductus endolymphaticus except a general growth in size, accom-
panied by a movement towards the brain, so that it comes to lie in
close contact with this organ. As the distal part comes close to the
brain, it begins to expand and its duct narrows; at the same time the
upper lip of the duct elongates, so as to carry the vestibular opening
downwards. The distal enlarged part grows, and, as the tadpole
loses its tail, assumes the permanent proportions, becoming at the same
time thin walled and vascular, while the organs of the two sides meet
both above and below the brain. Whether actual communication is
set up is difficult to determine by means of sections alone. The growth
of cartilage between the expanded end of the organ and the rest of the
vestibule does not take place until late and even then a foramen is
left, through which the duct passes from the vestibule to the skull-
cavity. It would seem to have some function of importance in the
adult, as it steadily increases in size during the growth of the tadpole
.and it is after the tadpole stage is passed that this increase in size be-
comes most rapid and the blood supply most copious.

DESCRIPTION OF DEVELOPMENT
1. Material and methods of preparation

The material consisted of larvae of Rana temporaria Linné.
The investigation was conducted chiefly by means of series of
sections, of which thirty were made, six representing the first
stage and four each of the following stages. The larvae were
first narcotized with ether and the gut extracted. ‘The larvae
were then fixed in sublimate (twenty-four hours). As I wished
to pay special attention to the lime contents of the saccus, it was
not possible to decalcify. This rendered section cutting very
difficult. In order to make the preparations more fit for section-
ing, they were left a long time in the various media. ‘They re-
mained in 70 per cent alcohol for twenty-four hours, in 95 per
cent for thirty-six hours, and in 100 per cent for twelve hours.
Thereupon a little cedar oil was added to the 100 per cent alcohol,
~ and the quantity of oil was gradually increased. After four hours,
the larvae were placed in pure cedar oil, in which they remained
four weeks. At the end of this time they were brought once

236 BEATRICE WHITESIDE

more into 100 per cent alcohol (two hours) and then into xylol
(one hour). ‘They were then placed in a bath of xylol-paraffin
(two hours), in paraffin 40° (eighteen hours), and finally in
paraffin 58° (six hours). The best stains were obtained by a
combination of haemalum with picrie acid. Many larvae were
also prepared macrosopically.

Stage I (fig. 2)

The youngest larva that I examined was 4 mm. long (measured
from the tip of the head to the aperture of the anus). The mouth-
opening and internal gills had appeared.

The invagination and constricting off of the auditory sac had
also'taken place. The latter organ has now a somewhat spheri-
cal form, and, as in this stage there is no cartilaginous capsule,
it lies close to the side of the hind-brain, being separated from
this by a thin layer of mesoderm cells (fig. 2). On its medioven- -
tral side the ganglion acusticus (g.a.) lies between it and the
brain. It is divided into utriculus (wir.) and sacculus (sac.)
by a septum and already possesses the rudiments of the semicir-
cular canals, lagena, pars neglecta, and pars basilaris. Within
the auditory sac numerous lime otoliths can be seen.

The ductus endolymphaticus (d.e.) is a well-differentiated
canal, situated at the median side of the auditory sac. This
duct starts from the upper median part of the sacculus and runs
dorsally at the median side of the-utriculus, extending in a dorsal
direction even further than the latter organ. Its long axis thus
runs in a dorsoventral direction. In contrast to later stages,
it is large in comparison with the auditory sac.

At its upper end the ductus expands into a small vesicle,
whose diameter is about twice as large as that of the ductus
proper. This vesicle extends somewhat in a cranial direction.
It represents the rudiment of the saccus endolymphaticus (s.e.).

The histological structure of the ductus and saccus endolym-
phaticus at this stage, in contrast to later ones, is exactly alike.
This corresponds to the fact, established by Alexander (’90) and
confirmed by Fleissig (’08), that in the development of the laby-
rinth the histological differentiation sets in much later than the

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 237

fe.

ca.sc. Lat.

Fig.2 Transverse section through the head of a larva. (Stage I.) Laby-
rinth region. a, aorta br., gill; ch., chorda; d.e., ductus endolymphaticus; g.a.,
ganglion acusticum; h, heart; m, mouth; sac., sacculus; s.e., saccus endolymphat-
icus; wir., utriculus; ven. 4, fourth ventricle. Figures 2-9 drawn by means of
Edinger’s projecting apparatus. Leitz. obj. 3, oc. 3.

Fig. 3 Transverse section through the head of alarva. (Stage II.) Laby-
rinth region. c.t., connective tissue; ca., caleareous matter; f.e., foramen endo-
lymphaticum; /, labyrinth; n.d.m., neural lamella of dura mater; p.d.m., periosteal
lamella of dura mater; v.s., junction between neural and periosteal lamella of
dura mater.

238 BEATRICE WHITESIDE

morphological. At the start the whole structure is lined with a
one-layered epithelium of cylindrical cells.

For comparison, it can be noticed that Norris (92) found the
first indication of thesaccus endolymphaticus in alarva of Amblys-
toma of 9 mm. length, whose auditory organ, similar to the
above-described frog larva, already possessed the rudiments of
the semicircular canals and the lagena. Fleissig (08) mentions
the first appearance of the saccus endolymphaticus of Phyllo-
dactylus in an embryo 4 mm. long, at a time when the develop-
ment of the labyrinth proper is far advanced. In reference to the
origin of the saccus in man, Streeter (16) writes that this organ
appears at about the time of the closing off of the semicircular
canals.

Stage II (figs. 3 and 11)

Stage II is based on a larva of 10 mm. length. No indications
of extremities are visible.

The development of the labyrinth is far advanced and all its
morphological parts have appeared (fig. 38). <A cartilaginous
ear capsule is completely formed, in whose median surface the
foramen endolymphaticum (f.e.) is situated. In this stage as
well as in the two following ones, this opening lies lateral to the
anterior end of the plexus choroideus of the fourth ventricle.

The ductus endolymphaticus has now attained its definitive
form. Its long axis has changed its dorsoventral direction and
inclines somewhat in a craniocaudal direction, so that its entry
into the cranial cavity lies a little more caudad than its exit from
the sacculus. In consequence of this rotation, only its upper
part is to be seen in figure 3. The ductus at this stage begins
with a slightly broadened piece at the upper median part of the
sacculus, and runs dorsally, median to the utriculus as far as the
foramen endolymphaticum. Here it turns inward and leads
through this aperture into the cranial cavity, where it immediately
expands into the saccus endolymphaticus. As a comparison of
figures 2 and 3 shows, the ductus endolymphaticus now appears
smaller in proportion to the rest of the labyrinth, which has
grown considerably more than the ductus.

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 239

The saccus endolymphaticus, though larger than in stage I,
still remains, in contrast to the labyrinth and ductus, in a
rudimentary stage, and does not arrive at the complicated form
which it has in the adult animal until much later. It lies on the
roof of the fourth ventricle (fig. 3, vent. 4) extending a little
eranially and caudally beyond the foramen endolymphaticum.
Its lumen is largest in the region of this opening and tapers
slightly cranially and caudally.

The position of the saccus endolymphaticus in relation to the
spinal meninges has been much discussed. In the first place,
the descriptions of these membranes have not been uniform.
Hasse (’73) and Rex (’93) mention three meninges: 1) the dura
mater, which closely invests the bones; 2) the arachnoidea,
separated from the dura by the subdural space, and, 3) the pia,
closely enveloping the brain and the spinal cord. According
to the opinion of the above-named investigators, the saccus
endolymphaticus is situated in the subdural space between the
dura and the arachnoidea. Sterzi (’99) has a different view.
He terms the membrane immediately investing the bone, the
endorhachis, and says that ventral to this les the dura mater,
whereas the innermost membrane is the arachnoidea. Accord-
ing to Sterzi, the saccus endolymphaticus would lie between the
endorhachis and the dura mater, separated from the endorhachis
by the epicaleary space, and from the dura by the epidural
space. Gaupp (04), Coggi (90), and O’Neil (98) describe the
membranes as follows: 1) the dura mater, which is divided dor-
sal and lateral to the brain and the spinal cord into two lamellae,
one of which closely invests the bone, while the other lies more
ventrally, and, 2) a primary vascular membrane, in which a pia
and an arachnoidea are as yet not distinctly differentiated. The
lymphatic space between the two lamellae of the dura is called
the interdural space, and that between the inner lamella of the
dura and the vascular membrane the subdural space. Accord-
ing to these observers, the saccus endolymphaticus lies in the
interdural space. My investigations confirm this view in every
respect (figs. 3, 4, ect.p.d.m. and n.d.m.). As O’Neil describes
in detail and I was able to verify, the dura mater is divided in

240 BEATRICE WHITESIDE

the whole region of the saccus endolymphaticus into two lamellae,
which unite ventral to that structure. The saccus endolym-
phaticus is closely connected with the periosteal lamella of the
dura, whereas it is joined loosely, if at all, to the neural lamella.
Above the plexus of the fourth ventricle a union of the saccus
endolymphaticus and the neural lamella of the dura takes place
with the primary vascular membrane. O’Neil’ proved that
the division of the dura into two membranes is much less marked
in the salamander in consequence of the much smaller expansion
of the saccus endolymphaticus.

As ean be seen in figure 3, there is at this stage a difference
between the histological structures of the ductus and saccus
endolymphaticus. Both organs are lined with a one-layered
epithelium, but the walls of the ductus are thicker than those of
the saccus. They consist of cylindrical cells whose nuclei lie
in the center. The walls of the saccus are composed of low plate-
epithelium cells, which, in transverse sections, show polygonal
dividing lines. These cells have their large oval nuclei in the
center. Many dark pigment granules are found in the proto-
plasmic part of the cells. The saccus is surrounded by a connec-
tive-tissue membrane.

In this stage the saccus endolymphaticus still appears as an
undivided sac, and has not yet the character of a gland. In
spite of this fact, I found many lime erystals within the sac.
This indicates that the cells are able to produce lime from the time
of their formation—a fact which will not surprise us, if we re-
member that these cells originally came from the labyrinth.

Stage III (figs. 4, 5, and 12)

Stage III corresponds to a larva of 11 mm. length, which does
not show any visible indication of extremities.

Figure 12 shows the extension of the saccus endolymphaticus.
Compared to stage II, there is only a simple increase in length
in a craniocaudal direction to be noticed. ‘The saccus now ex-
tends from the optic lobes to the end of the medulla oblongata.
The course of the anterior part is as follows: Starting from the

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 241

foramen endolymphaticum, it runs dorsal to the plexus of the
fourth ventricle into the region of the cerebellum. ‘There it
turns to the side and runs lateral to the brain as far as the anterior
end of the lobi optici, where it ends. Its largest voluminal
development is found in the region of the foramen endolymphati-
cum, where it is very broad, covering a large part of the roof of
the fourth ventricle. It narrows cranially. There is as yet no
connection between the saccus of the right side and that of the

- m.obl,
fa
* et:
“ch. <7 4
v. tse dt. tse.”

Fig.4 Transverse section through the head of a larva. (Stage III.) Pos-
terior part of the medulla oblongata. m.obl., medulla oblongata.

Fig.5 Transverse section through the head ofa larva. (Stage III.) Laby-
rinth region. d.t., point where saccus endolymphaticus divides; f.s.e., tubuli of
saccus endolymphaticus; v, blood vessel.

left. The part of the saccus which is situated behind the foramen
endolymphaticum lies dorsal to the brain. It tapers in a caudal
direction to a still larger degree than was the case in its anterior
part. Here, too, the saccus of the one side is separated from
that of the other (fig. 4), and there is no indication of the joining
of the two sacci, such as occurs some time later in the region of
the posterior end of the fourth ventricle (compare fig. 7).

The histological differentiation shows no further development
than the preceding stages. The saccus is lined with the typical

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 2

242 BEATRICE WHITESIDE

epithelium. However, this organ no longer represents an undi-
vided sac, but now ee in some parts of two parallel tubuli
(fig. 5). This is brought about by division of the formerly
single sac. The division does not take place in any fixed region,
but may apparently occur in any part. ‘There is great variability
in this respect in the different larvae which I examined. The
only thing which could be ascertained definitely in regard to the
region in which the division of the saccus occurs, is the fact that
the division never takes place within the region of the foramen
endolymphaticum. In the specimen described here as stage
III, the left saecus endolymphaticus is divided a little behind the
foramen endolymphaticum into two parallel tubuli running from
the front to the back (fig. 5). The median one has the greater
diameter. These tubuli run a short distance toward the back and
then join again. There is a similar division in the anterior part
of the right saccus endolymphaticus, in the region of the lobi
optici. Not only is the region in which the saccus divides not
always the same, but the manner of division also varies. Some-
times the tubuli are formed by invagination and adhesion of the
dorsal and ventral walls of the saccus, in other cases the invagina-
tion proceeds from the ventral wall only and continues until
it reaches the dorsal side (fig. 5, d.t.). All parts of the saccus
endolymphaticus are filled with calcareous matter.

Stage IV (figs. 6, 13,and 17)

A larva of 12 mm. length corresponds to stage IV. No trace

of limbs is apparent.

The saccus endolymphaticus extends fore the hemispheres —
into the region of the seventh vertebra (fig. 13). This stage
is particularly interesting because several subdivisions of the
saeccus can now be distinguished. Starting from the foramen
endolymphaticum, the pars anterior of the main stem ‘runs, as
in the preceding stage, dorsal to the brain as far as the lobi
optici. Here it fastens itself to the lateral circumference of the
middle brain, expands a little in the niche between the labyrinth
and the eye and reaches the posterior part of the hemispheres.
At its cranial end it sends out a process which ascends obliquely

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 243

ae a aire. —.

6 L g. fac. g: ae | ap

SEE :
pt aa:

Fig.6 Transverse section through the head ofa larva. (Stage IV.) Hypoph-
ysis region. g.fac., ganglion faciale; g.trig., ganglion trigemini; hyp., hypoph-
ysis; l.opt., lobi optici; s.e., saccus endolymphaticus.

Fig.7 Transverse section through the head of the larva. (Stage V.) Posterior
part ofmedullaoblongata. n.d.m., neurallamella of dura mater; p.d.m., periosteal
lamella of dura mater; ¢.s.e., tubuli of saccus endolymphaticus; v.s., junction
between neural and perisoteal lamellae of dura mater; v.sp.d., vena spinalis dor-
salis duralis.

244 BEATRICE WHITESIDE

upwards and inwards. In the middorsal line the ascending
processes join and unite with the paraphysis: This connection
between the two sacci, called by Gaupp the processus ascendens
anterior, has now attained its definitive form.

The pars posterior of the main stem of the saccus endolym-
phaticus extends from the foramen endolymphaticum into the
region of the seventh vertebra. ‘The saccus of the one side is
still separated from that of the other. The lumen of the saccus
decreases caudally.

The lateral extension of the saccus endolymphaticus is illus-
trated in figure 17. As mentioned above, it lies at the side of
the hemispheres, the diencephalon and the lobi optici. It is
narrow in the region of the hemispheres and the diencephalon;
in the region of the lobi optici, however, it widens and fastens
itself onto the dorsal side of the ganglion prooticum commune
(fig. 6, g.pr.c.). In the adult frog this ganglion represents
the union of the trigeminal and facial ganglia. At the stage
described here the two components are still to be recognized, the
facial ganglion (g.fsc.) lying dorsal to that of the trigeminus
(g.trig.).

In the larva taken to represent this stage of development,
the anterior part of each saccus endolymphaticus is an undivided
sac. The posterior part of each saccus is, however, divided into
two tubuli directly behind the foramen endolymphaticum.
These tubuli run as far as the posterior end of the fourth ven-
tricle, where they join once more. The whole saccus is filled
with lime.

Stage V (figs. 7, 14, and 18)

The sections on which the descriptions of stage V is based
were made from a larva 15 mm. long, in which the anterior
extremities had appeared.

The first thing to be noticed is that the position of the foramen
endolymphaticum has changed in its relation to the brain. This
opening no longer lies beside the plexus of the fourth ventricle,
but at the side of the cerebellum. The change in position is
probably due to the fact that the cerebellum has expanded
backwards in the course of its rather late development.

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 245

The dorsal extension of the saccus endolymphaticus is illus-
trated in figure 14. In its anterior part only an increase in size
can be observed. In the posterior part, however, a great change
has taken place. At the end of the fourth ventricle the sacci
endolymphatici of the two sides grow towards each other and
meet in the median line (fig. 7). From the point of juncture the
united sacci run caudally as far as the seventh vertebra.

Figure 18 shows the lateral expansion of the saccus endolym-
phaticus. In this stage the processus ventralis can be seen
(fig. 18, pr.vent.). It starts from that part of the saccus stem
which hes lateral to the lobi optici and dorsal to the ganglion
prooticum commune. The processus extends ventrally as far
as the lateral surface of this ganglion.

In the region of the foramen endolymphaticum the saccus
endolymphaticus is an undivided sac. Directly in front of and
behind this aperture, however, it divides into several smaller
tubul. In the cranial part four tubuli are formed, which run
independently of one another into the region of the hemispheres,
where they coalesce. The caudal part of the saccus remains
undivided to the end of the fourth ventricle. At the place where
the sacci endolymphatici of the two sides join, each saccus divides
into two tubuli (fig. 7, t.s.e.) which run parallel to each other,
and, towards their ends, split up into many small tubuli. The
division sometimes takes place in a horizontal direction, some-
times in a vertical one.

Each of the tubuli is lined with the characteristic epithelium
of the saccus endolymphaticus and is embedded in a delicate
structure of connective tissue. The tubuli are surrounded by
many blood vessels. Between the two partes spinales runs the
vena spinalis dorsalis duralis (fig. 7, v.sp.d.). At the place
where the two partes spinales separate, this vena divides into
two branches, which run toward the front in the outer walls of
the sacci and finally lead into the foramen trigemini. Both the
stem and the two branches collect all the veins of the saccus.

The lumen of the saccus endolymphaticus is filled with cal-
careous matter.

246 BEATRICE WHITESIDE
Stage VI (figs. 8, 15, and 19)

The animal representing this stage was going through meta-
morphosis. It was 15 mm. long and possessed well-developed
anterior and posterior extremities. The tail was somewhat
reabsorbed.

The foramen endolymphaticum now lies still farther forward
in relation to the brain. It is situated lateral to the lobi optici,
in which position it is also to be found in the adult frog.

In the posterior part of the saccus endolymphaticus a new
communication appears between the two sacci. It is formed by
the processus ascendens posterior, which, in the region of the
cerebellum, runs over the brain and connects the saccus of the
right side with that of the left (fig. 15, pr.asc.post.).

The most striking difference between this stage and the pre-
ceding one is the appearance of the calcareous sacs on the spinal
ganglia. These structures are formed in the following manner:
The posterior stem of the saccus, which runs backward inside
of the vertebral canal, sends forth processes which extend through
all the intervertebral foramina and as far as the spinal ganglia.
The number and state of development of these transverse proc-
esses vary in the different larvae examined at this stage. Some
specimens have many processes, others very few. The condi-
tion illustrated in figure 15 can frequently be observed. Here
all the processes (y) have appeared, although in different degrees
of development. Some of the sacs cover the entire ganglion,
others only its proximal part. The most cranial processes,
however, always appear first.

The processus ventralis has now completed its growth and
extends over the lateral and posterior surfaces of the hypophysis
(figs. 19 and 8). Behind the hypophysis it runs underneath the
brain until it joins the processus of the opposite side in the mid-
ventral line. In this way a connection between the two sacci -
endolymphatici takes place beneath the brain as well as above it.

The division of the saccus into small tubuli has progressed
further, and the whole organ now consists of many such structures.
This continuous division was observed in a general way by Coggi

oh

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS DAT,

9

Fig. 8 Transverse section through the head of alarva. (Stage VI.) Hypoph-
_ysisregion. d.t., point where saccus endolymphaticus divides; g.pr.c., ganglion
prooticum commune; f.s.e., tubuli of saccus endolymphaticus; v., blood vessel.

Fig.9 Transverse section through spinal cord and 2 spinal ganglia of a young
frog. (Stage VII.) g.spin., spinal ganglion; r.d., dorsal root of spinal nerve;
t.s.e., tubuli of saccus endolymphaticus; v.sp.d., vena spinalis dorsalis duralis;
z., enlarged as fig. 10.

248 BEATRICE WHITESIDE

(90), who examined older larvae and adult frogs. He writes
as follows:

Nei girini di rana le varie cavita che formano il sacco endolinfatico
sono molto meno numerose e meno suddivisi che non aceada nelle rane

adulte, ove la suddivisione va tant’ oltra gino a formare gli otricelli
microscopici che ho descritto.

Stage VII (figs. 9, 10, and 16)

This stage is based on a young frog which had just completed
metamorphosis.

Compared with stage VI, the only change in the appearance
of the saccus endolymphaticus is the increased size of the
caleareous sacs. These structures have now assumed their
definitive form. With this step the development of the saccus
endolymphaticus is finished. The organ now has the following
expansion.

Soon after the ductus endolymphaticus enters the cranial
cavity through the foramen endolymphaticum, it expands into
a large thin-walled sac, which lies in the interdural space, lateral
and dorsal to the brain and the spinal cord. From the foramen
endolymphaticum a part of the saccus runs cranially, lateral to
the lobi optici and the diencephalon, and reaches the anterior
lateral surface of the hemispheres. ‘Two processes start from this
main section of the pars cranialis anterior. From its anterior end
the processus ascendens anterior runs dorsally until it meets
the corresponding process of the other side. In the region of
the foramen endolymphaticum the processus ventralis runs ven-
trally and surrounds the ganglion prooticum commune and the
lateral surface of the hypophysis. Behind the latter organ it
proceeds farther in a median direction until a union of the proc-
esses of the two sides takes place.

The pars posterior starts from the foramen endolymphaticum
and runs caudally, lateral to the lobi optici and the cerebellum,
and dorsal to the plexus choroideus of the fourth ventricle.
Above the cerebellum the narrow processus ascendens posterior
connects the two saecci. At the end of the fourth ventricle the
pars posterior widens in a median direction and the sacci of the

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 249

*& a 10

Fig.10 Transverse section of one of the spinal ganglia and calcareous sacs
shown in fig. 9. c.tf., connective tissue; ca., caleareous matter; g., ganglion cells;
n.d.m., neural lamella of dura mater; ¢.s.e., tubuli of saccus endolymphaticus;
v., blood vessels.

Figure 10 drawn by means of Zeiss Abbe drawing apparatus. Oil immersion
O,3 mm. comp. oc. 6.

250 BEATRICE WHITESIDE

two sides unite in the middorsal line. From this point the two
sacci run together in a caudal direction, giving the appearance of
an unpaired structure. They leave the cranial cavity through
the foramen occipitale and extend inside the canalis vertebralis
to the region of the seventh vertebra, where the two halves part
again. ‘Through each intervertebral foramen processes emerge
which envelop the spinal ganglia. The extreme posterior end
of each saccus is formed by the calcareous sacs on the spinal
ganglia IX and X.

The above description corresponds with the one given by Gaupp
(07) for the adult frog. Other investigators have maintained
that the expansion of the saccus endolymphaticus varies in
different specimens. Gaupp explains this as follows:

Die einzelnen Teile des Saccus endolymphaticus sind nur dann gut
zu erkennen, wenn sie mit der charakteristischen milchweissen Fluessig-
keit gefuellt sind. Der Fuellungszustand wechselt aber, und so kann
es leicht kommen, dass einzelne Teile nicht sichtbar sind. Dies mag
wohl der Grund sein, wenn der Saccus gelegentlich eine geringere Aus-
dehnung zu besitzen scheint.

I should like to emphasize the fact that, in the larvae and very
young frogs, some parts of the saccus are invariably quite full
of calcareous matter, whereas other parts contain only a very
small amount. The greatest accumulation is always to be
found in that part of the saccus which surrounds the ganglion
prooticum commune and the hypophysis and in the part which
lies on the roof of the fourth ventricle. The partes spinales and
the calcareous sacs contain a fair amount, while I could observe
only very few crystals in the anterior cranial part and in the
processus ascendens anterior and posterior.

The differentiation of the saccus endolymphaticus into small
tubuli has progressed so far that each part of that very extensive
organ consists of many such structures. They are so numerous
that a description of each one would lead us too far. Some facts,
however, must be noticed. In the region of the foramen endolym-
phaticum, the saccus is an undivided sac, which, however, imme-
diately in front and in back of this aperture, divides into many

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS Zyl

tubul. These again split up into still smaller ones. In the
main part of the saccus all the tubuli run in a ecraniocaudal
direction. ‘The processus ventralis consists of four or five com-
paratively large tubuli running in a dorsoventral direction and
coalescing in several places (fig. 8). The partes spinales and the

12

Figs. 11 to 19 Diagrams of the central nervous system and labyrinth (Sac-
cus endolymphaticus black). Spinal ganglia not drawn in figs. 11 to 14.

Fig. 11 Dorsal view of larva. (Stage II.) d.e., ductus endolymphaticus;
l, labyrinth; sac., sacculus; s.e., saccus endolymphaticus; wtr., utriculus; ven. 4,
fourth ventricle.

Fig. 12 Dorsal view of larva (Stage III.) Jl.opt., lobi optici.

calcareous sacs are composed of a large number of tubuli. In
each pars spinalis there are three of these structures. Figure 9
represents a transverse section in the region of an interverte-
bral foramen. I found that the tubuli of one-half of the partes
spinales never join with those of the other half. The connec-
tion between the partes posteriores of the two sacci endolym-

252 BEATRICE WHITESIDE

phatici is thus only an external one. Between the two lies the
vena spinalis dorsalis duralis.

The calcareous sacs (fig. 10) have been described by Len-
hossék (86). My results confirm his statements, except for
his description of the position of these structures. According

pr. ase. an.

pr asc. an.
hem.cer._,

13

Vig.13 Dorsal view oflarva (Stage IV.) dien., diencephalon; hem. cer.,
hemisphaerae cerebri; pr. asc. an., proscessus ascendens anterior; p. spin., partes
spinales of saccus endolymphaticus; sp.c., spinal cord.

Fig. 14 Dorsal view of larva. (Stage V.)

to him, they cover only the proximal part of the ganglion, whereas
my sections show that they envelop the whole ganglion. Len-
hossék writes that the sacs lie inside the fibrous capsule belong-
ing to the ganglia. This capsule sends septa of connective
tissue into the interior of the sacs. There is also a thin continua-

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 2993

tion of the capsule, which runs between the sacs and the ganglia.
I can mention one more important fact, namely, that no nerves
or nerve-endings are to be found either in the calcareous sacs
or in any part of the saccus endolymphaticus. The whole
organ consists merely of glandular tubuli embedded in connec-
tive tissue.

pr.asc.an.

1S

Fig.15 Dorsal view of larva. (Stage VI.) g. spin., spinal ganglia; par.,
paraphysis; pr.asc.post., processus ascendens posterior; y., transverse processes
in different stages of development.

The above-mentioned investigator also gives a very proper
account of the finer structure of the tubuli of the calcareous
sacs, which I found to hold good for the tubuli lying within the
other parts of the saccus endolymphaticus. He states that
these tubuli are lined with a single-layered epithelium, whose
cells, in transverse section, are almost square and measure about
14 or 15y in diameter. Some of the cells, however, are cylindri-

254. BEATRICE WHITESIDE

eal in form, while others are very flattened. He thinks this
difference in shape is due to the fact that some of the tubuli are
quite full of calcareous matter, whereas others contain very little.
The cells have sharp, distinct outlines and oval nuclei. ‘To this
description I may add that the nucleus of the cells is very large
and contains no nucleolus. The chromatin appears in the form

pr. asc.ant.

16

Fig.16 Dorsal view of young frog. (Stage VII.) (Only right saccus endo-
lymphaticus drawn.) z., transverse processes in definitive form.

of large lumps—a condition which reminds one of that inthenuclei
of glandular cells. The protoplasm also recalls that found in
such organs, showing very fine granulation and containing some
pigment globules. Lenhossék was not able to prove the exist-
ence of a membrana propria around the individual tubuli. This
structure was found by Coggi (’90).

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS ZO
The lumen of the tubuli is filled with a liquid in which many
lime crystals are suspended. Carus (’41) was the first to notice

the similarity of these crystals to those in the labyrinth proper.
He writes as follows:

l.opt. ven4

dien: ‘g.prc. Spice

pe vent.

Sp. (oh,

Lateral view of larva. (Stage IV.) g. pr. c., ganglion prooticum com-

Fig. 18 Lateral view of larva. (Stage V.)

pr. vent., processus ventralis.
Fig. 19 Lateral view of larva. (Stage VI.)

256 BEATRICE WHITESIDE

Si l’on ouvre de bas en haut le labyrinthe d’une Grenouille, on est
surpris de trouver le petit sac plein d’une masse crétacée presque entiére-
ment de méme nature que les singuliers corps laiteux ou crayeux qul
garnissent les trous intervertébraux destinés au passage de nerfs rachi-
diénes. Les deux masses quand on les examine au microscope, parais-
sent consister en plusieurs millions de cristaux de carbonate calcaire,
arrondis et ovalaires, dont les plus gros ont environ un centiéme de
ligne de long, et dont la forme est celle d’un prisme a six pans terminé
par des sommets a six faces.

The entire saccus endolymphaticus may, as Lenhossék suggests
in regard to the calcareous sacs, be compared to a tubular gland
without an outlet. The contents of the saccus would then repre-
sent the secretion of the glandular cells. In any case, we must
not forget that these cells are derived from the labyrinth, whose
epithelium has the same ability to produce lime.

Summary

The first fact to be noticed in the course of development of the
saccus endolymphaticus is the comparatively late differentiation
of this organ. Ina larva of 4mm. length, whose auditory organ
is in an advanced state of development, the saccus endolymphati-
cus is present only as a shght expansion of the distal end of the
ductus. At a time when all the morphological parts of the
labyrinth are recognizable, this structure is still a small sac,
adhering to the roof of the fourth ventricle. According to
Norris (92) and Fleissig (08), the first indication of the saccus
appears also very late in Amblystoma and Ascalabotes.

The further development of the saccus proceeds very slowly.
First an increase in length takes place in a craniocaudal direction,
until the saccus reaches from the hemispheres into the region of
the seventh vertebra, the saccus of one side remaining separated,
however, from that of the opposite side. Next there develops,
in a larva 12 mm. long, the processus ascendens anterior. ‘This
is very soon followed by the joining of the partes spinales and
the first indication of the processus ventralis. The processus
ascendens posterior appears at the beginning of the metamor-
phosis. About this time the calcareous sacs also are to be seen.

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS ZO

These last-mentioned structures attain their definitive form at
the end of the metamorphosis.

At the time of its first appearance, the saccus endolymphati-
cus is an undivided sac lined by a single layer of epithelium. It
soon becomes partitioned into two tubuli which later subdivide
into smaller ones. During the course of development, the saccus
is divided more and more into small tubuli until it finally has
the appearance of a glandular structure.

The histological structure of the cells lining the saccus endolym-
phaticus remains practically the same during the whole period
of development. ‘The cells are first cylindrical in shape, later
they are cuboidal. Blood vessels are present at first only in
small numbers, but later become very numerous. The calcareous
contents of the saccus exist almost from the very beginning.

Throughout the entire course of development, no part of
the saccus endolymphaticus suffers retrogression, its develop-
ment is a continuous and direct one. Its development also
proceeds very slowly. For these reasons, it is certain that
the saccus endolymphaticus of the frog does not represent a
larval organ.

REMARKS ON THE STRUCTURE OF THE DUCTUS AND SACCUS
ENDOLYMPHATICUS IN THE VERTEBRATA

Since the investigations of Hasse (’73) and Retzius (’81),
no comparative-anatomical study of the ductus and saccus
endolymphaticus has been made, although some very interesting
papers on this organ in individual Vertebrata have been pub-
lished. Unfortunately, not all vertebrate types have been
examined in regard to this particular structure. It is, however,
possible to draw some conclusions from the facts hitherto ascer-
tained. Therefore, I shall give a short description of the anatomi-
cal structure of these organs, based on the above-mentioned
works and the more recent reports.

Hasse writes:

Saemtliche Wirbeltiere besitzen eine aus dem Vestibulum sich erhe-

bende Roehre, die, mit Ausnahme der Plagiostoma, wo dieselbe auf
die Schaedeloberflaeche fuehrt, bei allen Tieren in die Schaedelhoehle

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 2

258 BEATRICE WHITESIDE

sich begibt. Es ist dies der Ductus endolymphaticus oder Aquaeductus
vestibuli mit dem Saccus endolymphaticus von dem wir wissen, dass
es eine blind geschlossene Ausstuelpung des Labyrinthblaeschens
gegen das Cavum cranii hin darstellt.

Hasse found this structure in Myxine and Petromyzon. In
these animals the organ starts from the inner ear, or saccus com-
munis, goes through the apertura aquaeductus vestibuli, and
ends in the cavum cranii with a slight enlargement. The dilated
end is filled with calcareous matter.

The Elasmobranchii have been investigated by Hasse and
Retzius. In these animals the ductus endolymphaticus is a
very noticeable structure opening to the exterior. In Chimaera
it runs from the sacculus almost straight to the top of the head.
In the sharks it expands just beneath the opening, into a saccus,
either a small one as in Scyllium or a somewhat larger one as in
Acanthias and Aquatina. ‘This last-named animal is, in this
respect, a transition form leading to Raja, in which the ductus
dilates and forms a large sac lying almost horizontally under the
skin. Trygon and Torpedo have a formation similar to that
in the sharks. In all cases the ductus as well as the saccus is
filled with lime crystals which run out when the skin is pressed
in the neighborhood of the opening. This is also the case in
living animals.

Retzius examined five Ganoidei, namely, Acipenser, Lepi-
dosteus, Amia, Polypterus, and Calamoichthys. In Acipenser
the ductus endolymphaticus runs upward from the sacculus and
expands beside the upper end of the utriculus into an oblong
vesicle which adheres to the dura mater. In Lepidosteus and
Amia the relations are similar. Polypterus and Calamoichthys,
which are particularly interesting on account of their relation-
ship to the Crossopterygii, do not deviate either.

The. question whether a small duct, which in the Teleostei
starts from the sacculus and leads upward a short distance only
to end blindly without an enlargement, is to be looked upon as
the ductus endolymphaticus has been much discussed. Krause
(01) denies this and founds his opinion on the manner of develop-
ment. Wiedersheim (’09) agrees with him. On the other hand,

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 259

Fleissig (08), Wenig (11), and Keibel (15) think it probable
that this duct is rightly called the ductus endolymphaticus.
This structure is not found in the Siluridae, Cobidae, Cyprinidae,
Percidae, and Clupeidae.

The anatomical structure of the recessus labyrinthi in the
Dipnoi is very interesting. Retzius was unable to find this
organ, as he examined badly preserved specimens. It is now
known that the ductus and saccus endolymphaticus of Neocera-
todus resemble the same structures in the Ganoidei. Burne
(13) writes as follows:

The saccus endolymphaticus is a capacious pear-shaped vesicle with
bluntly rounded apex, situated to the mesial side of the space enclosed
by the anterior semicircular canal, with its apex inclined somewhat
forward. It is supported by a sheet of membrane (? dura mater)
within which wedged in between the apices of the two sacci endolym-
phatici is a large vessel, probably a vein. The lower end of the saccus
endolymphaticus bends slightly forward along the sinus anterior utri-
culi and gradually narrows to form the ductus endolymphaticus which
crosses the utricle near its anterior end and opens by a funnel shaped
mouth into the anterior extremity of the sacculus. These organs show
a great resemblance to the same organs figured by Retzius in the
sturgeon.

Protopterus, which was examined by Burckhardt, (’93), differs
greatly from the above-described animal. The saccus endolym-
phaticus is here an inflated bag lying in the cavum cranii and
giving rise to many tube-like diverticule which are filled with
calcareous matter. The organ covers nearly the whole of the
sinus rhomboidalis and extends caudally as far as the root of
the first pair of spinal nerves. ‘The saccus of the one side does
not communicate with that of the other.

The first description of the conditions in the Urodela was pub-
lished by Calori (50). He found between the bulbae auditoriae
of the axolotl a sac containing calcareous matter, extending
from the lobi optici over the corpora quadrigemina and the
medulla oblongata. He connected this structure with the
labyrinth, but did not explain the relations in detail. Hasse
proved that this organ represents the two sacci endolymphatici,
which are here amalgamated. In Triton, Hasse found condi-

260 BEATRICE WHITESIDE

tions similar to those in the axolotl, the sacci, however, being
separate. In salamander, he found that the sacci extend also
below the brain.

In Rana the relations are especially complicated, and accord-
ingly were explained much later. Here the ductus leads from
the sacculus into the cranial cavity, where it expands into a large
saccus. In contrast to the condition in the Urodela, this organ
is not confined to the cranial cavity, but extends in the verte-
bral canal as far as the seventh vertebra, and sends out processes
which come to lie on the spinal ganglia. I have given a detailed
account of the whole organ in the first part of this paper.

The different parts of the saccus endolymphaticus in Rana
were discovered separately. The calcareous sacs on the spinal
ganglia were necessarily the first to attract attention on account
of their conspicuous position. They were found by Blasius in
1681. Hasse (’73) discovered a chalky mass in the cavum cranii,
which he identified as the sacci endolymphatici. Finally Cogegi
(90) proved that the sacs on the spinal ganglia are outgrowths
from the main stem of the saccus.

In most of the Anura, examined in regard to this organ, the
relations are similar. Sterzi (’99) described the saccus in Rana
temporaria, ,Rana esculenta, Bufo vulgaris, Bufo viridis, and
Hyla arborea. In both species of Rana he found the above-
described expansion of the organ. In Bufo and Hyla there is
only a slight deviation, in so far as the processes of the spinal
part merely penetrate into the intervertebral foramina, but do
not extend beyond these apertures. Coggi (’90) states that
Discoglossus pictus and Bombinator igneus do not possess a
spinal part of the saccus endolymphaticus. He does not describe
the cranial part. Rex (’93) also examined Bombinator igneus.
In this animal he found a thin vascular membrane lying on the
roof of the fourth ventricle. This structure he takes for the
degenerated saccus. In Pelobates, this author found the same
expansion of the saccus as is known in Bufo.

In the Reptilia the ductus endolymphaticus is found in its
typical development. The saccus, however, is very small, and
contains lime in the embryo only. Carus (’45), who was the

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 261

first to examine the saccus endolymphaticus in this class, writes
that in Coluber natrix the saccus is represented by a small vesicle
lying directly under the suture between the parietale and occi-
pitale. The sacci of the two sides lie close to one another, but
do not join. Hasse investigated Anguis fragilis, Lacerta viridis,
Chelonia midas, Testudo graeca, and Crocodilus. In all of
these animals he found the expansion of the saccus similar to
that in Coluber.

The only reptiles whose saccus endolymphaticus does not
conform to the above-given description are the Ascalabotae.
According to Wiedersheim’s (’76) investigations on Phyllodacty-
lus, the saccus does not only run upwards to the roof of the brain,
but also extends backwards. It leaves the cranial cavity through
a small aperture in the parietale, passes back between the muscles
of the neck, and ends in the region of the pectoral girdle with
a large closed sac. All the parts of the saccus are filled with
calcareous matter, in the adult as well as in the embryo. In
Phyllodactylus the two sacci do not join; in Ascalabotes, however,
they coalesce a little behind the posterior part of the parietal
suture. They separate again when leaving the cranial cavity.
Many species of Platydactylus have a similar extension of the
saccus. Gray (’08) did not find this formation in Tarentola
mauretanica. He thinks that perhaps it was destroyed by the
manner of preparation.

Hasse writes that the structure of the recessus labyrinthi in
Aves is very similar to that in the Reptilia. He was not able to
state whether lime crystals are present at any period of develop-
ment. According to Wiedersheim (’06), the saccus is filled with
lime in the embryo stage.

The Mammalia show a slight deviation in the anatomical
structure of the ductus endolymphaticus. In these animals the
duct arises from the labyrinth with two branches, one from the
lower median side of the utriculus, the other from the upper
median part of the sacculus. The two branches soon join and
the ductus leads upward at the median side of the utriculus, as
far as the foramen endolymphaticum. Here it runs into the
cranial cavity and ends inside the dura mater with a small dila-

262 BEATRICE WHITESIDE

tation. Here, too, according to Wiedersheim, the saccus con-
- tains lime in the embryonic stage.

If we compare the anatomical structure and the relative size
of the saccus endolymphaticus in the various classes of Verte-
brata, we see the following interesting course of development.
The saccus is present in all Vertebrata, at first as a slight dilata-
tion at the end of the ductus, then as a large vesicle, and finally
in the Anura and Ascalabotae as an enormous sac lying in the
cavum cranii and the vertebral canal (respectively, the shoulder-
region). From this maximum of development, the size of the
saccus begins to decrease and at last returns in the Mammalia
to a hardly perceptible enlargement at the end of the ductus.

The above-given summary is incomplete because the number
of animals investigated in regard to the saccus endolymphaticus
is small. Furthermore, the groups that have been compared do
not represent a natural phylogenetic line of evolution. There
are, however, some interesting facts which may perhaps be con-
nected with some systematic problems.

Let us first examine the group of the Dipnoi. Here we find
that Neoceratodus possesses a ductus and saccus endolymphati-
cus which are very similar to the corresponding structures in
Polypterus. Thus we can add a new fact to the many points
of similarity mentioned by Huxley (’76) and Dollo (’95) between
the Dipnoi and the Crossopterygii. This is particularly inter-
esting because many investigators, as Huxley, Parker (’92),
Dean (’92), etc., who took their facts from Retzius, described
the auditory organ of the Dipnoi as similar to that of the Selachii.

If, on the other hand, we wish to consider the relationship be-
tween the Dipnoi and the Amphibia, we find that the recessus
labyrinthi of Protopterus is very similar in structure to that of
Rana.

The large saccus in Protopterus has probably developed from
the more simply constructed one of Neoceratodus through spe-
cialization, due, one might assume, to adaptation to terrestrial
life. Protopterus is generally considered to be a much more
specialized form than Neoceratodus. Thus Parker (’92) pointed
out that in Protopterus the reduction of the gills has proceeded

DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 263

much further than in Neoceratodus. We also find in Protop-
terus two pulmonary sacs, whereas Neoceratodus possesses
only one. Parker writes of Protopterus:

‘Although the lungs are usually said to be paired in their
origin, the outgrowth which forms them, though bifurcating
close to its origin, is in fact so far as I can gather unpaired at —
the first.”

This fact might also support the view that Neoceratodus is
the more primitive animal. The uniserial fin of Protopterus
has been regarded as a form developed from the biserial fin of
Neoceratodus. Thus it seems as if the relationship between
Neoceratodus and Protopterus indicated by the structure of the
recessus labyrinthi is in reality a true one.

As may be expected from their systematic relationship, all
the higher vertebrates, reptiles, birds, and mammals, have a
similarly constructed saccus endolymphaticus. The exceptional
position of the Ascalabotae appears therefore very strange. In
these animals, as we have seen, the saccus leaves the cranial
cavity and runs between the muscles of the neck into the shoul-
der region, where it ends as a large closed vesicle. As there can
be no thought of a close relationship between Ascalabotae and
Anura, this similarity (at least as far as size is concerned) of the
extension of the saccus is probably due to convergence. There
is no question here of a convergence caused by a similar habitus,
as the Ascalabotae are entirely terrestrial animals, many of
them even living in deserts. Such a convergence could only be
explained as having arisen, in spite of a different mode of life,
through the fact that the stimulus which causes the great exten-
sion of the saccus is the same from a mechanical point of view.
This question can only be answered by a knowledge of the func-
tion of the organ. As far as I know, no experiments to ascer-
tain the function of this structure have been made.

Several theories as to its probable function have been advanced.
Hasse (’73) thought this organ served to regulate the pressure
in the labyrinth by sucking up the endolymph out of the saccu-
lus, when the pressure there was too high. Wiedersheim (’76),
Keibel (’15) and Streeter (’16) have adopted this theory. Hasse

264: BEATRICE WHITESIDE

added that in the case of animals possessing a very large saccus
this organ might transmit the sound waves from the skull into
the ear. Wiedersheim thought this also highly probable. Sterzi
maintained that the saccus in Rana had the same function as the
other spinal meninges, namely, that of protecting the spinal
cord. Carus (’41), who observed the calcareous matter in the
saccus of snake embryos and the subsequent disappearance of
the same, concluded that the lime was used for the growth of
bone and was thus absorbed. Gaupp (’96) has accepted this
theory in regard to Rana. In this case, the growth of bone took
place in the adult frog as well as in the larva. Therefore, the
saccus secreted lime throughout the life of the frog.

Scott and Euclid Avenues,
St. Louis, Mo.

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DEVELOPMENT OF THE SACCUS ENDOLYMPHATICUS 265

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THE AMERICAN JOURNAL OF ANATOMY
vou. 30, No. 3, MAy, 1922

Resumen por el autor, Herbert G. Willson.
Las terminaciones del bronquiolo humano.

El autor ha hecho dos reconstrucciones negativas en cera, con
un aumento de 100 didmetros, una de un pulmén de un adulto
y la otra del pulmén de un nifio. Ambos modelos representan
un bronquiolo respiratorio y sus ramas, habiéndose trazado
algunas de estas hasta su terminacién. Algunas de las con-
clusiones mds importantes son las siguientes: 1) En las ramifica-
ciones existe mayor complejidad, irregularidad y mayor entre-
lazamiento que lo que se ha supuesto generalmente. 2) La
ramificacién es dicotémica hasta que llega a las terminaciones,
transformandose después en irregular, si bien con tendencia a
la economia de espacio. 3) No existe espacio esférico o atrio,
conforme ha descrito Miller. 4) No existen comunicaciones
interalveolares directas. 5) El pulmén del nifo es tan com-
plicado en estructura como el del adulto. 6) En el pulmén
adulto, durante la inspiracién profunda oridinaria e drea total
de epitelio respiratorio y no respiratoro no es mayor de 70
metros.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MARCH 27

THE TERMINALS OF THE HUMAN BRONCHIOLE

HERBERT G. WILLSON

University of Toronto

NINE FIGURES

In the hope of throwing some light on certain questions about
which there has been controversy, the construction of a wax
model of a respiratory bronchiole was begun at the University
of Toronto in October, 1919. The work was carried out in
collaboration with Prof. J. Playfair MeMurrich, by whom the
problem had been suggested and to whom the writer is very
greatly indebted for advice and assistance.

The extreme complexity of the terminal branches of the bron-
chial tree is not generally appreciated. The maze of channels
which occur even in a minute piece of lung tissue cannot be
visualized accurately from a mere comparison of serial sections.
The larger passages of the lung may be injected with wax or
metal and a cast obtained by corroding away the lung tissue,
but one cannot be certain of obtaining in this way a complete
cast of the smaller tubes. The method of wax reconstruction
of serial sections is the only plan by which one may hope to get
clear ideas regarding the finer tubes, and even this method is
especially difficult to apply to the bronchioles. So complicated
are the branchings and so carefully has nature economized space
in the lung, that if all the air passages in a piece of lung tissue
are reconstructed in wax on a magnified scale, the result is prac-
tically a solid, and the model has to be dissected in order to show
the relationships of tubes and air-cells.

Malpighi in 1661 demonstrated the vesicular nature of lung
tissue and showed how the trachea terminates in bronchial fila-
ments, but after his time there was no important contribution
to the knowledge of the histology of the lung until the early part

267

268 HERBERT G. WILLSON

of the nineteenth century, when Soemmering, Rossignol, Reis-
seisen, and others published the results of their researches, and
Henle, by his discoveries in general histology, laid the founda-
tion for many special investigations. Controversy now arose
in regard to such questions as the exact shape of the terminal
bronchioles, their method of branching, and as to whether or
not there were direct communications between alveoli.

Rossignol, writing in 1847, refers to the most distal divisions
of the bronchial tree as ‘infundibula,’ and these he describes as
being thickly beset with alveoli. He notes that the alveoli of
the infundibulum are of an unusually: great depth, and that while
the alveoli are scattered and few in the proximal part of the
respiratory bronchiole, they are soon arranged close together,
covering the whole surface of the last bronchial divisions. As
to the method of branching, he concludes that there are both
dichotomous and trichotomous divisions. In his investigations
Rossignol inflated and dried the lung, after having injected the
blood vessels.

In 1860 Waters described monopodial, dichotomous and trichot-
omous branching. His conclusions were based on the study of
single sections. In man he found no alveoli in the terminal
bronchiole, but only in the infundibulum. He states that at a
certain place the terminal bronchiole widens into a cavity into
which open six, eight, or ten canals, beset with alveoli. These
canals he terms air-sacs, these being again identical with Rossig-
nol’s infundibula.

I. E. Schulze in 1871 used the term ‘Alveolengang’ to denote
all the parts of the tubular system on which there are alveoli,
excepting, however, the terminal sacs, for which he employed
the term infundibula.

In 1892 W. 8. Miller announced the discovery of a new ele-
ment in the series of pulmonary air-spaces, terming it the ‘atrium’
and locating it between the air-sacs (infundibula) and the ter-
minal bronchiole (alveolengang). This space seems to be identi-
cal with the enlargement of the terminal bronchiole described
by Waters as giving origin to the air-saes, but Miller describes
it as something more than a mere enlargement, having a more

TERMINALS OF HUMAN BRONCHIOLE 269

or less spherical form with numerous alveoli on its walls and
giving origin to from two to five air-sacs. The lung of the dog
was used in Miller’s first investigation; but in 1900 and again in
1913 he published accounts of further researches, and maintained
that his description held good for lung of cat, ox, child, and
adult man.

In his article of 1900 he gives the following table of nomencla-
ture for the air-spaces of the lung:

W.S8S. MILLER B.N.A. SCHAFER SCHULZE KOLLIKER

Bronchus Bronchiolus Bronchial Alveolengang | Alveolengang
respiratorius tube
Terminal Ductuli Lobular
bronchiole alveolares bronchus
Atrium
Air-sac Air-sac Infundibu- Infundibu-
Tumi lum
Air-cell Alveolus Air-cell Alveolus Alveolus
pulmonis

But at the same time he revised his own earlier terminology,
substituting the B. N. A. terms for ‘Bronchus’ and ‘Terminal
bronchiole.’ In his investigations Miller used the method of
wax reconstruction. His results have found wide acceptance
by the authors of text-books, in spite of several dissenting voices.

In 1900 Justesen gave an account of his investigations of the
structure of the lung in oxen. He used corrosion preparations
and also serial sections, drawings of which were made on trans-
parent paper, so that by superposing the drawings, successive
sections might be compared. He finds that each ‘bronchiolus
simplex’ forms dichotomously two respiratory bronchioles, each
of which again divides dichotomously, each of the branches so
formed ending in a large cavity which he identifies with the
atrium of Miller. His atria are variable in size, sometimes quite
distinct, and sometimes only slight enlargements of the bron-
chioles, and while Miller finds two to five air-sacs on each atrium,
and Waters six to ten, Justesen believes that there are normally
four. Two first bud out and these then divide, so that each of
the four occupies a position corresponding to one of the angles

270 HERBERT G. WILLSON

at the base of a four-sided pyramid, the apical angle of which
is occupied by the atrium. In other words, the air-saes do not
arise as accidental growths from the atria, but are formed by
two successive dichotomies in planes at right angles. In the
adult ox he found occasionally but three air-sacs on an atrium—
a condition which he explains by supposing that in the case of
the primary air-sacs the secondary dichotomy had failed owing
to space exigency.

Justesen holds that the bronchial branchings occur in a definite
mathematical plan and are fundamentally dichotomies, but
that in the majority of the branches the dichotomy becomes modi-
fied into a sympodial arrangement, the terminal branches still
retaining the dichotomous plan. If this be so, and the mathe-
matical regularity of the dichotomies persist, the lateral branches
of each sympodial stem might be expected to show a decreas-
ing number of air-sacs as they were traced peripherally, one
arising from an earlier dichotomy having twice as many air-
sacs as that which arose from the succeeding dichotomy. Jus-
tesen believed that he obtained evidence in favor of this arrange-
ment in his observation on the pig where the eparterial
bronchus gave rise to as many lateral branches as did the stem
branches for the rest of the lung.

F. E. Schulze, writing in 1906, takes the view that Miller’s
atria are not new spaces, but only those parts of the ductuli
alveolares into which the sacculi open. He states:

So wenig, wie man an einem sich unregelmissig verzweigenden Baum-
ast diejenigen Stellen, wo sich ein Ast in zwei oder auch mehrere
Endiste teilt, als besondere typische Stellen charakterisieren und mit
einem eigenen Namen, sondern einfach als Teilungsstellen zu bezeichnen
pflegt, so wenig scheint mir in dem respiratorischen Gangsystem der
Lunge die Auszeichnung dieser Stellen durch eine besondere Benennung
(‘Atrium’) erforderlich oder auch nur zweckmiassig zu sein.

Schulze claims that normally in man and in many mammals
there are direct communications between alveoli—‘alveolar
pores’—and in this view he is supported by Hansemann, Has-
sall, Zimmerman, Nicolas, and Merkel, but is opposed by
Piersol, W. 8. Miller, Laguesse, and Oppel.

TERMINALS OF HUMAN BRONCHIOLE 201

In 1907 J. Miiller investigated the lungs of most of the domes-
tic animals, using metal corrosions as well as sections. His
conclusions regarding the occurrence of atria he states as follows:

Hinsichtlich des neuen Luftraumes, des Atriums, war es mir nun
weder an den Korrosionspraparaten noch an den Schnitten bei irgend
einem unserer Haussiiugetiere méglich, ihn alseinen Luftraum sui gene-
ris bestitigen. Wenn auch da und dort einmal ein Alveolengang vor
seiner Auflésung in die Infundibula eine buchtige Erweiterung zeigte,
welche etwa dem ‘Atrium’ Justesens entsprechen kénnte, so habe ich
doch niemals zwischen jedem Infundibulum und dem Alveolargang,
noch auch zwischen mehreren Infundibeln und einem solchen einen oder
mehrere kugelige Hohlriume eingeschaltet gesehen, welche fiir das kon-
stante Vorkommen der Millerschen Atrien sprechen kénnten.

Miiller found alveolar pores in various animals, but not in
young animals. He thinks these are pathological.

Just as my first model was completed, I received the number
of The American Journal of Anatomy that contained two arti-
cles by the Japanese investigator Ogawa, who by an interesting
coincidence had been working in the University of Kyoto at
exactly the same problem as myself and by similar methods,
but had evidently begun the construction of his model some
months before I started with mine. Ogawa worked with human
material, and constructed both a negative and a positive model
of the terminal branchings of the lung, the former being at a
magnification of 100 diameters and measuring 8 x 12 x 8 cm.,
while the latter was enlarged 80 diameters and measured 11.3
x 24x 20cm. Like Schulze and Miller, he reaches the con-
clusion that ‘‘ Miller’s atrium is an unnecessary term, at least for
the human lung.” In his second paper he states that ‘‘alveolar
pores are normally found in many mammals, and only seldom
cannot be seen.’’ Further reference to Ogawa’s work will be
made when his results are compared with my own.

MATERIAL

The material used was exclusively human and consisted of
portions of the lungs of two individuals obtained at autopsies
performed soon after death. One of the individuals was a woman
of thirty years who had died of heart disease (mitral stenosis),

Dia HERBERT G. WILLSON

while the other was a child whose age could not be definitely
ascertained, but was certainly less than thirteen years. In the
ease of the adult, portions of suitable size were taken from the
lungs and immersed in Bouin’s fluid, but in the case of the child’s
lung the entire organ was first injected through the bronchus
under gentle pressure with Bouin’s fluid, and then immersed in
the same fluid, portions suitable for sectioning being taken only
after the tissue had been fixed in this manner. The portions
selected were carried through the various grades of aleohol and
imbedded in paraffin, and to secure satisfactory penetration
of the paraffin they were, while in 70 per cent alcohol, placed
under the bell-jar of an air-pump and the air exhausted till
bubbles ceased to rise from the cut surface of the tissue.

By this method a perfect infiltration of the paraffin was ob-
tained, and the tissue was cut into serial sections, 204 thick in
the case of the adult lung and 30. in that of the child. Both
series were stained with Weigert’s elastic tissue stain, this being
chosen with the intention of studying later the distribution of the
elastic fibers in the human lung. Wax reconstructions of the air
spaces, ie., negative reconstructions of portions of each lung
were made at a magnification of 100. To ensure accuracy in the
superposition of the wax plates in the model of the adult, numer-
ous bridges were left in cutting out the air-spaces, but in the
second model the necessary accuracy was obtained by the use of
a duplicate series of drawings of the sections made upon trans-
parent paper. A duplicate drawing of a section about the middle
of the series was covered by a sheet of glass, and on this the pieces
of wax representing the corresponding air-spaces were placed
one after the other, as they were cut from the wax plate. The
next succeeding drawing was then carefully oriented upon that
first chosen, so that the position of the air-spaces shown in the
one could be accurately determined with reference to those of the
other, and from the information thus obtained the pieces of
wax representing the air-spaces of the second section could be
accurately adjusted on those cut from the first plate. Dealing
in this way with successive drawings and wax plates, half the
model was built up. This completed portion was then detached

TERMINALS OF HUMAN BRONCHIOLE Die

from the sheet of glass, turned upside down, and the other por-
tion of the model was then built up in the same way. This
method of orientation was found to be much more economical of
time than was the use of bridges and entailed no sacrifice of
accuracy.

To follow a respiratory bronchiole from its beginning to its
terminals, it was necessary to, make drawings from 128 sections
of the adult lung. As each section was 20 thick, it is evident
that the piece of lung containing all these branches had a thick-
ness of 2560 un, 1.e., 2.56 mm. or a little over 1/10 of an inch.
It will be evident also that the height of the completed model
would be 256 mm., or a little over 10 inches. In the case of the
child’s lung, drawings of sixty-three sections were required, and
as each section was 30 » thick, the thickness of the piece of lung
reconstructed was 1890y, or 1.89 mm., and the height of the
completed model approximately 7.5 inches.

RESULTS

The first impression received from inspection of the completed
models is that the branchings of a respiratory bronchiole are far
more complicated than is revealed in the text-books, and one
feels also that there is difficulty in ‘labeling’ the various parts
according to the terms commonly used. In some places a num-
ber of alveoli are represented in the reconstruction as opening
into a cavity which seems too small to deserve the name of an
air-sac, while in another place one finds an alveolus which is
several times as large as the ordinary alveolus. The models
indicate that the minute passages in the lung are not formed in
strict accordance with the usual descriptions. The two models
when placed side by side suggest at once that the child’s lung is
a miniature of the adult lung, just as the child’s hand is a minia-
ture of the adult hand, there being no apparent difference in
complexity of structure. More air-sacs occur in the volume of
child’s lung represented than in the greater volume of adult lung
represented in the first model.

A photograph of the model from the adult lung is shown in
figure 1. It starts with a non-respiratory bronchiole which is

aie h HERBERT G. WILLSON

marked 3a, and is so designated because in tracing it back through
the serial sections it was found to represent the third dichotomy
from a bronchus which contained cartilage in its wall. The
3a dichotomizes into branchings marked 4a and 4b, of which
4b has not been followed any further, but 4a again divides dichot-
omously into Ja and 5b, whose walls show alveolar outbranch-
ings, so that they are to be regarded as respiratory bronchioles.
The 56 is followed only a short distance, but 5a again divides into
two stems, one of which was followed for some distance, but its
reconstruction is omitted in the photograph for the sake of sim-
plicity. The other stem, which may be designated 6a, is com-
pletely reconstructed, and gives rise to all that portion of the
model which is colored. It can be followed into a further dichot-
omy, one branch of which gives rise to the portions colored
orange and green, while from the other all the remaining por-
tions originate. The orange and green portions have been
separated from the rest of the model in order that its parts might
be more completely shown.

A photograph of the model on this scale, though useful for a
general orientation of its parts, does not sufficiently reveal the
details. ‘These are more clearly shown in figure 2, which repre-
sents a part of the portion colored orange in figure 1 at a greater
magnification. It shows a number of infundibula or air-saes
with their alveolar outbranchings, and it shows also how difficult
it is to determine exactly what shall be termed an air-sac and
what an air-cell. Thus the lower of the two portions colored
yellow might equally well be regarded as a single air-sac with a
number of complicated air-cells, or as at least two air-sacs with a
common basal portion. Similarly, the upper yellow portion
might be regarded as a single large air-sac or as three, according
to the point of view of the observer. The terms infundibulum
or air-sac (ductulus alveolaris B. N. A.) and alveolus or air-cell
are all useful in conveying an idea as to the arrangement of the
terminal air-spaces of the lung, but it must be remembered that
in the human lung, at least, transitions exist between them;
particular cases may be found where it is difficult to say whether
one is dealing with an air-sac or an air-cell.

TERMINALS OF HUMAN BRONCHIOLE - AAAS)

To obtain a clearer picture of the terminal branchings, those
represented in both models were projected upon a single plane,
the projections being based partly on tracings of various sections
used in the construction of the models, and partly on sketches
of the smaller parts. The result obtained in the case of the
model of the child’s lung is shown in figure 3. The stem marked
1 is a non-respiratory bronchiole which was traced through sev-
enty-four sections (2.22 mm.) to reach its origin from a bron-
chiole with cartilage in its wall. Four dichotomies occurred in
this distance. In the model of the adult lung three dichotomies
occurred between the first respiratory bronchioles and the bron-
chiole with cartilage in its wall. The stem (1) divides into two
branches, only one of which (2) was followed; this was a respira-
tory bronchiole, alveoli occurring on its wall. It in turn under-
goes a dichotomy, only one limb of which (3) was followed, and
then two additional dichotomies succeed in rapid succession,
only one branch of each being followed. That followed from the
last of these dichotomies (6) again divides into 7 and 8, these
again into 9 and 10 and 11 and 12, respectively, but beyond this
the branchings become irregular, and while it would be possible
to interpret some of these divisions as dichotomies, there are
others where the branching could be more accurately termed a
trichotomy. In fact, the branching in some parts is so irregular
that almost any ‘method’ might be read into it. The truth seems
to be that, as the terminals are approached, no one system of
branching is followed, but one edict is obeyed, i.e., that there
must be no waste of space.

Embryological investigation has shown that in the early devel-
opment the branching is dichotomous, and apparently this is
continued, with some modification in certain of the branches,
until there comes a time when the small bronchioles are competing
with one another for space, and then they branch or send out
processes in any possible direction. This competition for space
of the infundibula and alveoli, seen in the complicated inter-
digitation of these elements from different respiratory bronchioles
and in their varying form and size, is the most striking impression
that one receives from a study of the models. An idea of the

276 HERBERT G. WILLSON

manner in which the infundibula fit in with one another may be
obtained from figure 4, which is a photograph of the pleural sur-
face of the model of child’s lung. The numbers on the infundib-
ula correspond with those on the branchings shown in figure 3.

From the diagram and the accompanying photographs it
will be evident that the models reveal no definite space which
corresponds to Miller’s atrium. Careful study of the models
shows, it is true, enlargements of the respiratory bronchioles
where several infundibula communicate with them, but these
enlargements exhibit no definite delimitation from the remaining
portions of the bronchioles, and they never assume a spherical
form. Schulze was probably correct in his contention that a
special name is not needed for that part of a branch from which
a number of subordinate branches arise.

It is interesting to note that Justesen’s description of the
branchings of a respiratory bronchiole applies very closely to the
branchings revealed by the models, except in regard to the atrium.
Both Waters and Justesen held very decided views as to the
planes in which successive branchings occur. Waters believed
the plane of two diverging branches to be always at right angles
to the plane of the two branches preceding. Justesen claims
that ‘‘there is a strong tendency of the dichotomous divisions
to lie in alternating planes cutting one another at right angles,”
but this was not universal. Examination of the models indi-
cates that Waters’ rule is by no means constantly true. Four
angles which, according to Waters’ rule, would be 90°, were found
to be approximately 85°, 90°, 10°, bat 45°.

The number of biatdlintes that intervene between a non-
respiratory bronchiole and an air-sac was determined in seven
cases, and in three the air-sacs were reached at the fifth division,
in three at the sixth, and in one ease at the seventh. Ogawa
found from two to nine ramifications, with an average from four-
teen cases of 5.57. Laguesse found six or seven branchings.

The alveoli or air-cells on seven different air-sacs were counted,
the numbers being as follows: 22, 14, 16, 18, 12, 16, and 20,
giving an average of 16.8. Ogawa’s average is 11. However,
many of the air-sacs, as Justesen says, are bifurcated or deeply

TERMINALS OF HUMAN BRONCHIOLE QE.

indented, and a good deal depends on whether the subdivisions
are considered as separate air-sacs or not.

Calculating from the lengths of the tubes in the model the
following are the actual lengths of these tubes in the adult lung.

mm.
INK SSS Ree oan AH RH Ciao Boks Pian ont bOI OREO NS Moca DER Ea IOe 1G
INIG iS SEARS Tn 6 MERITS SGI ORO Gachc GEO IEe HERG Th OCR NES rep ROTERS retusa 0.8
TSO) Te RR a aS, oO tee ee 0.5
INO US aca oc era at rier ae to lar hrs teh a ea eae ea a 0.5
INI OE Hens clo Sie CieEEsG Di GRRE SRIIEIG Gia Old Gk Atak BIS ORSLE EOE IGE? CoO Mncee Ears tat 0.5
INO GRR Ceo ee ne ee rte ene ESE, OG SOOO. Ca OOS Sr semene 0.4
ISON Gy Oe ee eee A 8 a a See Ne tS Oe he ie er eer 0.2

The greatest and least diameters of the non-respiratory bron-
chioles represented in the same model were estimated as follows:

mm

UNOS Blo islet CO Oh OC RCE AED CRETE IC ORT Eee One ee une ry Ah ean ae 0.3 x 0.40
DIN eA a Per ale arene ps se kot ek ows rie teeter chem ceo tap eeneie aa eet OP3ec0825
INGE CY Deas & GE OE Oe Ue IIE rN Seas rite irae the Baa oa) OMe 0e22

Similar estimates in the case of the respiratory bronchioles
were:

mm.
INGA DARREN SOO RR Se & cisR eter trek Ue eee cL ee a EE 0.4 x 0.30
INIG)S EO Re crea 2 cic bo Dae AGE IIO ORE MES SRI tio co rR Rd Geto Gero ony 0.3 x 0.35
INIGS GEE SB ES OB ET OOS o DOOD OG At OC ERE DE ACTE Sen SOM StT aean Doe One xa0E 30
SINCE OID eveprcrcepetreae eves cr ean cree nia: «1 ncaa haat ore EN etots es wace ah opealars wore 0.4 x 0.30

Measurements of the greatest and least diameters of those
tubes into which the air-saes open gave the following results in
three cases,—the figures indicating the actual dimensions in the
lung:

0.3 mm. x 0.2 mm.

0.5 mm, x 0.3 mm.
0.4 mm. x 0.3 mm.

This gives an average of 0.4 mm. X 0.27 mm.

Ogawa found the average diameter of an alveolar duct to be
0.24 mm., and he quotes Kolliker’s estimate as 0.27 mm. and
that of Schulze as from 0.4 to 0.2 mm.

It will be seen from these measurements and from the illus-
trations that the bronchial tree in its finer ramifications by no
means shows a decrease in the diameter of successive branches

278 HERBERT G. WILLSON

towards the periphery. There is, indeed, sometimes an increase
even before the air-sacs are reached.

The air-sacs themselves show a great diversity of shape and
size and frequently they are recurrent. Figure 2 shows plainly
the tendency of the air-sacs to widen out to a greater diameter
than the bronchiole from which they arise. It was, no doubt,
this widening-out tendency which caused the early investigators
to use the term ‘infundibulum,’ though the air-sacs are not
funnel-shaped. Calculations of the actual size of eight air-sacs
gave the following results, the measurements being taken in
three dimensions:

0.4x0.8x0.4 mm.
0.3 x0.6x 0.3 mm.
0.7x0.4x0.3 mm.
0.6x0.3x0.4 mm.
1.0x0.4x0.5 mm.
0.3 x0.4x 0.3 mm.

OF5eEx0ksix O22 mnie
0.4 x 0.6 x 0.2 mm.

The air-sacs or alveoli, as shown in the model, vary greatly
in size and shape. The following estimates were made of the
actual diameters in three directions of the alveoli of the lung:
(All the measurements given above have reference to the adult
lung.)

0.05 x 0.06 x 0.07 mm.
0.08 x0.08 x 0.12 mm.
0.08 x0.10x0.13 mm.
0.06 x0.08 x 0.10 mm.
ONL x08 bsx7 0.20) mm:
0.08 x0.10x0.13 mm.
0.08 x0.05x0.15 mm.
0.08 x0.10x0.10 mm.
Average: 0.075 x 0.09 x 0.125 mm.
Extremes: 0.05 and 0.20 mm.

Ogawa in the case of a man of thirty-one years found an average
of 0.1 mm. for depth and breadth of an alveolus, and in the case
of a man of fifty-six years, his estimates are 0.15 mm. depth and
0.19 mm. breadth. Ogawa’s extreme estimates, counting both
his cases, are 0.04 and 0.21. It will be seen that many of the

TERMINALS OF HUMAN BRONCHIOLE 279

alveoli represented in the model are elongated to a greater ex-
tent than is usually described, though, as has been mentioned,
Rossignol noted that certain alveoli had unusual depth. The
models confirm the observations of Rossignol regarding alveoli.

In the construction of the models and in the examination of
the sections, careful search was made for evidence of interalveo-
lar communications, but no evidence of their existence was found.
The air-sacs interlock with wonderful closeness, so that there is
absolutely no waste of space, and because of this close interlock-
ing it sometimes requires great care to satisfy oneself of the ab-
sence of interalveolar communications, but in no case could such
communications be demonstrated.

THE AREA OF PULMONARY AIR-SPACES

During the course of this work the interesting question of the
total area of the respiratory air-spaces naturally suggested it-
self, and an attempt was made to answer it by estimating the
total area of the respiratory epithelium in a cubic millimeter of
lung tissue and then multiplying this by the total volume of the
lung, expressed in cubic millimeters. It is evident that such a
method can give only approximately the actual respiratory sur-
face in the lung, since it takes no account of the variations that
may occur in the size and number of the air-spaces in various
cubic millimeters of the lung, and it fails to make allowance for
the larger non-respiratory bronchioles and bronchi. Yet the
calculation seemed worth carrying out, as it promised, at least,
a maximum figure beyond which the total respiratory surface
could not possibly extend.

In order to estimate the area represented in one cubic milli-
meter of lung tissue, a square of 100 mm. side was marked out on
each of fifty successive drawings in the series of adult lung, the ~
squares being oriented so that the series of squares represented
successive sections of tissue. Since the sections were 20 yu thick,
the fifty squares together represented sections totaling 1 cu.
mm.in volume. The total perimeter of the various air-passages
in each of these squares was measured. This was done by trans-
ferring the drawings within each square to millimeter paper, and

280 HERBERT G. WILLSON

counting the number of millimeters in the perimeter of each air-
space in that square, and totalling the amount. The grand total
for the fifty squares amounted to 69346 mm. Since the magnifi-
cation was 100 diameters, the corresponding perimeter in the actual

=a mm. and if this be multiplied by the thick-

lung would be

the result will be nearly 14 sq. mm.

ness of the sections zt
(1000)
of respiratory surface in 1 cu. mm. of lung tissue.

The lung tissue used in this estimation was obtained after the
lungs had collapsed—the pleura having been opened. Vier-
ordt estimates the volume of the lungs in this condition to be
from 3005 to 3975 ecm. Taking the volume as the average of
these, 3400 ccm. or 3400000 cmm., the area of the walls of the
air-passages (respiratory and non-respiratory) is approximately

son x aa < 3400000 sq. mm., or about 47 square meters.

Now, the volume varies as the cube of like dimensions, while
the area varies as the square of like dimensions, so that the area
would not be doubled if the volume of the lung were doubled by
expansion of air-passages. According to Arnold, the volume
of the lung when fully inflated is 6805 cem., and Vierordt states
that the volume is 9521 ccm. ‘bei stirkster Fillung.’ For the
areas corresponding to these estimates the extreme limits might
fairly be placed at 70 and 90 square meters, respectively. Vier-
ordt’s estimate possibly refers to artificial inflation of the lungs
after death. For the volume of the lungs on deep inspiration,
Arnold’s estimate seems a. reasonable one, since 5500 cem., in
the case of the adult lung, is the approximate total volume of
complemental, tidal, supplemental, and residual air. We are
thus led to the conclusion that on ordinary deep inspiration the
total area of the respiratory and non-respiratory epithelium is
approximately 70 square meters, and the respiratory area alone
must be considerably less than this. In order to estimate how
much less, one would have to know the proportionate amount of
respiratory to non-respiratory epithelium in the air-passages,
and this is not known. |

TERMINALS OF HUMAN BRONCHIOLE 281

It might be pointed out that the method of multiplying the
perimeter by the thickness of the section is exact only in the case
of a tube of uniform diameter. ‘To illustrate, it is plain that tke
cylinder formed by a pile of coppers has an area on its curved
surface equal to the sum of the areas of the edges of the coppers,
while the area of the curved surface of a cone is really greater
than the total area of the edges of a number of dises of gradually
diminishing diameter piled up to represent a cone. Here, then,
is a source of error which tends towards making the result too
low, while errors of omission in the counting or tracing would
tend in the same direction. On the other hand, the cubic milli-
meter of lung tissue on which our calculation is based contained
only the finer branchings of air-passages, so that the result would
be accurate only if the whole lung were made up of such fine
branchings. This source of error, tending to make a too high
result, can hardly be canceled by the factors referred to above.
The figures given probably represent the extreme upper. limits
of area corresponding to the respective degrees of expansion.

In Hermann’s Handbuch der Physiologie there is given a cal-
culation by Zuntz of the area of the respiratory surface. Zuntz
assumes the average diameter of an alveolus to be 0.2 mm. when
the lung is moderately inflated, and the total air-space of the
lung to be 3400 to 3700 cem. He considers that at least 3000
cem. of this space is occupied by alveoli and infundibula. He
calculates the volume of an alveolus as though it were a sphere,
and arrives at the following result for volume and area of a
single alveolus:

Volume, 0.00414 ccm.
Area, 0.125+ sq.mm.

Reducing the 3000 eem. to cubic millimeters, he divides this
volume by the volume of a single alveolus, and reaches the con-
clusion that the number of alveoli in the lung is 725 million. On
the basis of this result and the estimated area of a single alveolus,
he concludes that the area of the respiratory surface is $0 square
meters, when the lung is inflated to a moderate extent.

Aeby used the same figures as Zuntz for volume and area of a
single alveolus, assuming the average alveolus to be of spherical

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 3

282 HERBERT G. WILLSON

form and to have a diameter of 0.2 mm. Since the volume of
such a sphere is 0.004+ emm., Aeby concludes that in a cubie
millimeter of lung tissue there would be 250 such alveoli, each
with an area of 0.125+ sq. mm., so that in a cubic millimeter
of lung tissue the total area would be 250 x .125 or 31.25 sq. mm.

Nicolas gives estimates of the different areas of respiratory
epithelium corresponding to the various degrees of expansion.
He considers the maximum volume of air which the lungs will
hold to be 4970 cem. in the average man. Huis statements are
based on calculations by Aeby:

Le nombre total des alvéoles est immense. Huschke l’avait évalué
4 1700 ou 1800 millions. Selon Aeby ce chiffre est beaucoup trop élevé.
D’aprés ses calculs chaque millimétre cube de poumon comprendrait
250 alvéoles représentant une surface de 31.2 muillimétres carrés. En
estimant le volume du poumon a4 1617 centimétres cubes chez l’homme
et a 1290 chez la femme, on obtiendrait chez le premier une somme totale
de 404 millions d’alvéoles et chez la seconde de 322 millions, (en chiffres
ronds). Cette quantité correspondrait 4 une surface de 50 4 40 métres
earrés pendant l’expiration forcée, de 79 (homme) 4 63 métres carrés
(femme) pendant l’état moyen de repos, et enfin de 129 (homme) 4 103
métres carrés (femme) lors d’une dilation compléte.

The result here given of 129 square meters is greatly in excess
of my maximum result, in spite of the fact that in the caleula-
tions of Aeby and Nicolas the figure representing the volume
of the lungs is smaller. The difference arises from the difference
in the estimate of the number of square millimeters of area per
cubic millimeter of lung tissue. Nicolas uses Aeby’s estimate of
31.2 sq. mm., while my estimate is 14 sq. mm. To obtain a
result of 31.2 sq. mm., according to my method of calculation,
the air-spaces cut in an area of 1 sq.mm. would have to be much
more numerous than those of any of my sections of adult lung.

F. E. Schulze also refers to Aeby’s calculations. After ex-
plaining that Aeby assumes the average diameter of an alveolus
to be 200 u, he states that he cannot agree with Aeby’s estimate
of the number of alveoli and corresponding total respiratory
surface. Schulze used his own estimate of the volume, 1500
cem., for the lungs of the average man. To get the volume of the
respiratory parenchyma he deducts 20 per cent, leaving 1200

TERMINALS OF HUMAN BRONCHIOLE 283

ecm. He takes the volume of an alveolus to be 200% cubic microns.
Reducing the 1200 cem. to cubic microns, he divides the volume
of a single alveolus into this total volume and arrives at the
conclusion that there are 150 million alveoli in the lung, thus:

12 << 10%

- = 150,000,000
293 x 106

He then calculates the area of an alveolus as 5 x 200? square
microns, and estimates the total respiratory surface as 5 xX
200? & 150,000,000 square microns, or 30 square meters.

The estimates of Schulze and Zuntz are much higher than mine
in proportion to the figures which they use for the volume of the
lung.

In our calculation it was found that in the adult, a cubie milli-
meter of lung tissue represented the following area of lining
of air passages:

69346 20

—— = 13.869 sq. mm., or nearly 14 sq.mm.
100 1000

Similar calculations were made of the corresponding area in
the child’s lung, and estimates were made also from sections of
emphysematous human lung and from the lung of an opossum.
The results are given below.

ADULT NORMAL CHILD MAN OF 61, EMPHYSEMATOUS OPOSSUM

14 sq.mm. (nearly) 19 sqa.mm. 6 sq.mm. to 8.713 sq.mm. 27 sq.mm.

|

Some of the tracings on which these calculations are based
are reproduced in figures 5 to 9.

In the child’s lung only a few typical sections were counted,
and only one reading was taken of the opossum lung.

In the emphysematous lung, the total perimeters of twenty-
five consecutive sections were counted, the sections being of
tissue near the pleura, though there were other parts, also near
the pleura, where the emphysema was much more marked.
The readings of the twenty-five squares gave a total of 21784
mm., an average of 871.3 mm., which corresponds in the actual

Kach figure represents a square millimeter of a section of lung
The figures were traced at the same
magnification (X 100) and reduced in reproduction to X 60. The double lines
represent blood vessels. Fig. 5, section of a child’s lung. Fig. 6, section of an
opossum lung. Tig. 7, section of an adult human lung.

284

Figs. 5 to 7
tissue, traced with a projection apparatus.

S) |
Figs. 8 and 9 Each figure represents a square millimeter of a section of
emphysematous (human) lung tissue, traced with projection apparatus. Both

figures were traced at a magnification of 100 and reduced in reproduction to X 60.
285

286 HERBERT G. WILLSON

lung tissue to an average perimeter of 8.713 mm. Using a more
direct method of calculation than previously, this average perim-
eter of 8.7138 mm. multiplied by 1 (millimeter) gives 8.713
sq.mm., the approximate area of lining epithelium in each cubic
millimeter of emphysematous lung. The readings of eight other
sections of emphysematous lung, taken from near the pleura,
gave a total of 4728 mm., or an average of 591-mm., corresponding
in the actual lung to 5.91 mm., or nearly 6 mm. This average
perimeter in 1 sq.mm. corresponds to an area of 6 sq.mm. per
cubic millimeter of lung tissue.

It will be seen that the average for all the readings of the em-
physematous lung indicates that a man with emphysema might
possibly have only half the normal amount of respiratory epithe-
lium per unit of lung volume.

CONCLUSIONS REGARDING THE HUMAN LUNG

1. In the branching of the respiratory bronchioles there is
far greater complexity, irregularity, and a greater degree of inter-
locking than is usually described.

2. There is no spherical space, or ‘atrium,’ such as has been
described by Miller.

3. The method of branching of the bronchioles is dichotomous
until the terminals are approached, and then the branching
becomes irregular.

4. Counting as the first branch, a respiratory bronchiole aris-
ing from a non-respiratory one, the air-sac is usually reached at
the fifth to seventh branch.

5. There are normally no direct communications between
adjacent alveoli.

6. The bronchioles do not decrease in diameter as the periph-
ery is approached, but remain of fairly uniform size until the
air-sacs are reached, and the air-sacs are, as a rule, of greater
diameter than the tubes from which they arise.

7. Waters’ rule, that the planes of successive dichotomies cut
one another at right angles, is only exceptionally confirmed.

8. The lung of the child is just as complex in structure as that
of the adult.

TERMINALS OF HUMAN BRONCHIOLE 287

9. It is calculated that during ordinary deep inspiration the
total area of respiratory and non-respiratory epithelium in-the
adult lung is not greater than 70 square meters.

BIBLIOGRAPHY

Appy, Cur. 1880 Der Bronchialbaum der Siugetiere und des Menschen.

Henute 1873 Handbuch der Anatomie des Menschen, Bd. 2.

JusTEseEN, P. Tu. 1900 Zur Entwickelung und Verzweigung des Bronchial-
baumes der Siugethierlunge. Arch. f. mikr. Anatomie, Bd. 56.

Mitier, W. 8. 1892 The lobule of the lung and its blood vessels. Anat.
Anz., Bd. 7.
1893 The structure of the lung. Jour. Morph., vol. 8.
1900 Das Lungenlaippchen, seine Blut und Lymphgefiisse. Archiv. f.
Anat. u. Physiol., Anat. Abt.
1902 Article ‘Anatomy of the lungs.’ Reference Handbook of the
Medical Sciences, New York.
1907 A criticism of some of the recent literature on the structure of the
lung. Anat. Rec.
1913 The air spaces in the lung of the cat. Jour. Morph., vol. 24.

Miuier, J. 1907 Zur vergleichenden Histologie der Lungen unserer Haus-
siugetiere. Archiv. f. mikr. Anatomie u. Entw., Bd. 69.

Nicouas, A. 1903 Appareil respiratoire in Poirier’s ‘Traité d’ Anatomie humaine.’
Paris, T. 4, Fase. 2.

McLeop, J. J. R. 1920 Physiology and biochemistry in modern medicine,
3rd edition.

Ocawa, C. 1920 The finer ramifications of the human lung. Am. Jour. Anat.,
vol. 27, no. 3.
1920 Contributions to the histology of the respiratory spaces of the
vertebrate lungs. Am. Jour. Anat., vol. 27, no. 3.

Scuuuze, F. E. 1906. Beitrige zur Anatomie der Siugethierlungen. Sitzungs-
bericht kgl. preuss. Akademie d. Wiss.

VierorptT, H. 1906 Daten und Tabellen fiir Mediziner. Jena.

Zuntz, N. 1882 Hermann’s Handbuch der Physiologie, [V Band Theil. S. 90.

PLATE 1
EXPLANATION OF FIGURE

1 Reconstruction from an adult lung. This model was made at a magnifica-
tion of 100 diameters. The distance in the model from the top of the part colored
orange to the bottom of the part colored green is 7 inches.

TERMINALS OF HUMAN BRONCHIOLE PLATE 1
HERBERT G. WILLSON

289

PLATE 2
EXPLANATION OF FIGURE

2 Part of a reconstruction of an adult lung, more highly magnified. This
part is the right half of the portion colored orange in plate 1, seen from above.
The illustration shows the part at its actual size in the model, which was con-
structed at magnification of 100 diameters.

290

TERMINALS OF HUMAN BRONCHIOLE PLATE 2
HERBERT G. WILLSON

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fesumen por el autor, Edgar Allen
El ciclo éstrico del rat6n.

El autor compara las observaciones llevadas a cabo en
animales vivos con los cambios histol6gicos en el tracto genital
y los ovarios. Estos cambios son de naturaleza ciclica y re-
quieren un periodo medio de cuatro a seis dias. Los estados
estin representados por quilescencia, crecimiento, el climax
éstrico y degeneracién. Se manifiestan en la vagina por
la formacion ciclica y la degeneracién de una capa cornea.
Los cambos degenerativos en el epitelio uterino no siguen a
su. extirpacién; como consecuencia de esto la hemorragia
uterina es rara. La extrusién de los ntcleos en el epitelio ciliado
del oviducto es paralela a las fases degenerativas del utero y la
vagina.

En le ovario existen grandes foliculos durante la fase anabdélica
y son reemplazados por cuerpos amarillos en la fase catabdlica.
Por consiguiente, la ovulacién separa a estas fases y tiene lugar
durante el estro. La clasificacién del rat6n entre las especies
de ovulacién espontanea es errdnea. Algunos ovulan con
regularidad, otros tan solo de un modo esporddico, y otros tan
solo ovulan con un estimulo sexual adicional. Diferentes modos
en la longitud del ciclo son peculiares de diversas razas de
ratones. Puesto que el ciclo es corto y la ovulacén puede ser
espontdnea o no serlo, los ovarios pueden contener tres o cuatro
series de cuerpos amarillos voluminoses o no contener ninguno.
En ambos casos se presentan ciclos normales. A consecuencia
de esto parece jJustificada la conclusién de que los cuerpos amaril-
los del estro no poseen funcién causativa en los procesos
anabodlicos o catabdlicos del cico éstrico. Las pruebas acumu-
ladas indican la presencia de 6vulos en los foliculos grandes como
‘ausa del crecimento del tracto gental y el estro, y su ausencia
o atresia como la causa de la fase degenerativa.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MARCH 27

THE OESTROUS CYCLE IN THE MOUSE

EDGAR ALLEN
Department of Anatomy, Washington University School of Medicine

TWENTY-FOUR FIGURES

CONTENTS

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3. Points important in the anatomy of the genital organs of the mouse.... 303
4, External morphological changes during the oestrous cycle.............. 304
a. Anmna ls studied Histologically i)... 42g. cise slate eee eltin swe ti ton Do telteine bo 306
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waLimererations of the cycle sf2. vai. ht. ie. ote n oletalee eat eet evldse she 327
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PPPS LER OU URE CLUE sta aria iicss05, afm ovasstelee arate eh. © ein Se nila ola ache aS oikya We Gis oe 346

1. INTRODUCTION AND LITERATURE

Although the mouse, Mus musculus, has been used for em-
bryological purposes for nearly a century, no exact knowledge
of its oestrous cycle is available. One reason for this is that the
mouse, like other rodents, comes into ‘heat’ and receives the
male within from six to twenty-four hours after parturition, and
most investigators have timed their collection of embryological
material from this ‘heat? moment. Another reason for our lack
of knowledge .of the oestrous cycle in this form is the fact that
external signs of ‘heat’ have been relied upon for diagnosis. Con-
cerning this, Heape states: ‘‘It is difficult to determine the length
of the prooestrum and oestrus in rodents, since the external
signs which characterize these conditions are comparatively

297

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, No. 3

298 EDGAR ALLEN

slight.”’ In many animals I have found them to be either entirely
absent or so slight as to make an accurate diagnosis impossible.

Discussing the sexual phenomena of rodents, Marshall says:
‘‘Mus decumanus and M. musculus are known to experience a
recurrence of the dioestrous cycle for more than nine months of
the year in the absence of the male.’”’ The other three months
are probably winter. No observations on the length of this
cycle are given.

Sobotta (95) is the only embryologist supposedly timing
his stages from an oestrous period other than that following
parturition, and he believed the duration of the cycle to be
equal to the gestation period, i.e., 20+ days. Apparently all
writers up until as late as 1916-17 have followed Sobotta in
this particular.

Danforth in unpublished observations in 1914-15 attempted
to check the length of the cycle by tabulating the number of
days between two successive litters and computing the greatest
common divisor of these intervals. In his records of sixty-six
animals, twenty-three were excluded because of the possible
complication due to the mother lactating while pregnant. The
greatest common divisor of the modes of his curve was found to
be 4 to 6 days. This evidence is inconclusive, but it at least
casts doubt on the existence of a uniform oestrous cycle of 20+
days. .

H. P. Smith (17) attempted to approach the problem from a
consideration of the ovarian cycle. This method permits of
only one reliable observation, other than that of parturition, on
one animal, namely, that made on histological examination of
the ovaries after death. Conclusions must be drawn from a
series of animals on each of which only one observation is made.
The individual variation among even litter mates is so great as
to make this method very inaccurate although a large number of
animals be used. By this method Smith was led to conclude
that the oestrous cycle is one of great variability, averaging
seventeen and one-half days. He began the collection of his
series at parturition, and his results are therefore really the
recovery time of the ovaries—the time required for a resumption

THE OESTROUS CYCLE IN THE MOUSE 299

of ovulation after the interference of pregnancy. The variation
in this period is known to be great even within a single species,
therefore the conclusions drawn can scarcely be applied to the
normal ovarian cycle.

Estimates of previous oestrous periods arrived at from histo-
logical comparisons of the corpora lutea are not reliable since,
as will be shown in the present paper, many mice when isolated
from males do not ovulate spontaneously during oestrus.

Daniels (10) and King (13) have shown, in the mouse and
rat, respectively, that if an animal becomes pregnant at the
oestrous period following parturition and suckles her litter as
well, the gestation period of her second litter may be lengthened,
in some cases to from twenty-four to thirty days, an increase of
20 to 50 per cent. Kirkham (’16—’17) showed this delay to be
due to a failure of the embryos to implant in the uterine mucosa.
The embryos apparently remain free in the uterus in a state of
inhibited growth for a time equal to the extension of the gesta-
tion period. No histological differences have been reported
between the uterine mucosa of pregnant mice and those preg-
nant and also lactating. Long and Evans (’20), working on the
rat, have reported histological difference between corpora lutea
under these two conditions and (’21) a limiting influence of com-
bined pregnancy and lactation on the growth of the vaginal
epithelium. For several reasons, therefore, it seems better to
time embryological material from a mating during a heat period
not immediately preceded by parturition.

Further literature on the oestrous cycle in the mouse is limited
to isolated observations made incidentally during investigations
of other problems.

Concerning the literature on other rodents Marshall (’10) ac-
cepts Heape’s statement that ‘‘dioestrous cycles recur for five or
six months in the domestic rabbit, and that if oestrus is ex-
perienced in winter it may occur independently of the possibility
of pregnancy. While some animals exhibit oestrus every three
weeks fairly regularly, others do so every ten days; on the whole,
I think 10-15 days is the usual length of their dioestrous cycle.”

300 EDGAR ALLEN

This is the sort of observation on which so much of the literature
of oestrus is based. .

Lataste has done considerable careful work on the vaginal
plug of rodents and in this connection notes that the dioestrous
cycle is usually about ten days.

Loeb (’11 a) reported a sexual cycle in the guinea-pig recurring
every twenty to twenty-five days and described the histologi-

cal changes in the uterus and ovaries at intervals. In a later
paper (711 b) he concludes the duration of the cycle to be from
fifteen to twenty days.

In 1917, Stockard and Papanicolaou described a method of
diagnosing the stages of oestrus in animals showing only slight
external signs of their condition by a histological examination
of the cell contents of the vaginal fluid. The cellular content of
this fluid changes characteristically as the cycle progresses.
This method offers the advantage of providing a complete his-
tory obtained from the repeated observation of reliable criteria
upon the same living animal. It thus permits the study of
individual variations and gives a record of the events taking
place before killing the animal to study histologically the internal
genital organs. By this method they obtained an oestrous cycle
in the guinea-pig of remarkable regularity averaging 16+ days.
This method also permitted Stockard and Papanicolaou to locate
very exactly the moment of ovulation in a living guinea-pig.
The rupture of the follicle occurs when the vaginal smear shows
a definite cellular picture.

In the rat, Heape placed the duration of the oestrous cycle at
ten days. Long and Evans (’20), using the above-mentioned
vaginal fluid examination method, have shown this to be from
four to six days, or actually only one-half as long as Heape sup-
posed.

Of course much valuable information concerning certain phases
of the sexual cycle in the mouse is already at hand in the work
of Sobotta, Kirkham, Long and Mark, Smith, and others; this
information, however, may be added to when approached from
another viewpoint.

THE OESTROUS CYCLE IN THE MOUSE 301

2. MATERIAL AND METHOD

Variation in the duration of the oestrous cycle has been reported
in nearly all mammals studied, not only in closely allied forms,
but within the limits of the same species. It seemed desirable,
therefore, to include some individuals from each strain of mice
in our colony. Several variations in other phenomena peculiar
to certain strains had formerly been noticed, the most striking
being the difference of reaction to ether anaesthesia: Albinos in
our stock are very susceptible, while brown and black are quite
resistant. It was thought important to find if there was any
variation in the oestrous cycle typical of different strains. The
stock chosen included brown, black, albino, dominant white
with black eyes, gray, agouti, and yellow, as well as hybrids of
these strains. More than ninety animals have been used in
this work, the majority being young virgin mice. A few who had
born litters were included for comparison, but in all cases records
were not begun until the animals had been separated from males
for at least a month after the removal of their young.

It has long been known that to maintain the oestrous rhythm
the animals must be healthy, well fed, and under uniform envi-
ronmental conditions. To eliminate crowding, no more than four
animals were placed in one cage. Cages had a floor space of
850 sq. cm. and a capacity of 17,000 ce.

A constant supply of water was accessible to the mice through
small holes in the ends of test tube containers. The food con-
sisted of oats, cracked corn, sunflower seed, and dog biscuits.

Usually a little hay was added as nest-building material.
During the winter the animals were kept in a heated room.

Methods

Stockard and Papanicolaou (’17) have shown the most reliable
criterion of the condition of heat in the guinea-pig to be the cell
content of the vaginal fluid. Their technique, slightly modified
because of the smaller size of the mouse, was followed. Some
animals were examined three times daily to get the exact time
relations of the various phases of the cell changes, but for the

302 EDGAR ALLEN

accumulation of data as to cycle length, examination once daily
was found to be adequate. The condition of the vulva, the
degree of opening of the vagina, and the nature of the vaginal
contents were noted. A histological examination of the smears
was always the deciding factor in the diagnosis. Smears were
made by the usual bacteriological technique. Haematoxylin
and aqueous eosin were used as stains. <A block with a converg-
ing trough which could be quickly covered was devised for
holding the animal while smears were being taken.

Animals used for correlating the conditions of different parts
of the genital tracks with each other and with the cellular changes
in the smears were examined and smears collected for several
cycles before they were killed. ‘These repeated examinations
gave some indication of their ‘degree of sexuality.’ Smears
were invariably taken immediately before killing. The animals
were killed instantly by a sharp rap at the base of the skull,
the abdominal cavity opened, and the conditions of the uterine
cornua noted as to size, transparency, blood supply, and con-
tractility. Shortly after the body cavity is opened, the uterus
contracts to such an extent that the marked differences present
at various stages in the cycle cannot be detected; therefore, an
immediate inspection is necessary. After examining the fresh
uterus the bladder was clipped off, the symphysis pubis sectioned,
the skin was cut around the vulva, and the phallus, vagina,
uterus, oviducts, and ovaries were dissected out and placed on a
glass slide. After a minute’s drying they adhere to the slide and
can be fixed without contortion. Bouin’s fluid was used as a
fixing reagent and proved satisfactory for both uteri and ovaries.
After hardening in the lower alcohols, the organs were cut
to facilitate orientation and imbedding. Serial sections 10.4
thick were made of the ovaries and oviducts. In trimming these
organs it is important that the peritoneal ovarian sac be left
intact to protect the surface of the ovary during dehydration
and imbedding. Several transverse serial sections of the middle
of each uterine cornu, the body of the uterus, the vagina, and the
cervix, as well as several serial sagittal sections through the
phallus and lower vagina, were cut at a thickness of 6 to 8u.

THE OESTROUS CYCLE IN THE MOUSE 303

Cut surfaces were oriented so that the same regions in different
animals could be compared (text fig. 1).

3. POINTS OF IMPORTANCE IN THE ANATOMY OF THE GENITAL
ORGANS OF THE MOUSE

There are some points in the anatomy of the genital organs of
the mouse which need emphasis since they have an important
bearing on certain aspects of this work.

Text fig. 1 Genital organs before fixation, showing the planes of histologica
sections. A, body of uterus; B, vagina; C, middle of left cornu of uterus; D
middle of right cornu; EH, left ovary and oviduct; F, right ovary and oviduct
H, sagittal section of phallus and lower vagina.

The vagina in the mouse opens directly on the vulva without
the protection of any structures homologous to the labia minora.
Its stratified epithelium is devoid of glands. The phallus is
large and prominent and is traversed in its entire length by the
urethra. The cervix is relatively short, the division into the
lumina of the uterine cornua occurring 1 to 3 mm. above the
opening of the external os. A stratified epithelium, similar,

304 EDGAR ALLEN

but considerably lower than that of the vagina, is continued up
into the cervix in virgins as well as in multiparae, but beginning
at the branching of the cervical canal, it becomes simple, non-
ciliated, cuboidal or columnar. Glands are uniformly distrib-
uted throughout the uterine mucosa except along the line of
attachment of its broad ‘mesentery-like’ ligament. The entrance
of the oviduct into the uterine cornu is surrounded by valve-
like folds of the mucosa, which make it very difficult to inject
the oviducts from the uterus. These valves are in a position to
guard against a back flow of fluid from the distended uterus into
the oviducts, which might be fatal to the passage of ova down
the tubes. The oviducts are ciliated only at the ovarian end
where the mucosa is thrown into high folds. Throughout the rest
of their extent the epithelium is simple columnar. The uterine
end can be distinguished from the middle segment by the low
folds of its mucosa, those in the middle segment being rela-
tively high.

The ovaries are situated just caudad to the kidneys and are
completely surrounded by closed sacs of peritoneum from which
the openings of the oviducts lead. Therefore, the number of
ova in the tubes and periovarian sacs is always the number
ovulated, since none can escape into the peritoneal cavity.

4, EXTERNAL MORPHOLOGICAL CHANGES DURING THE OESTROUS
CYCLE

a. Evidence from the external genitalia and cell contents of
the vagina. Among many rodents there is very little or no
discharge during ‘heat.’ The degree to which congestion and
reddening occur in the vulva is also variable, often being totally
absent or so slight as to be a very poor criterion of oestrus. In
a total of 355 cycles chosen at random to decide this point in
the mouse, only 190, or 53.5 per cent, showed well-marked ex-
ternal signs. And seventy-three, or 23.3 per cent, showed the
vulva and vagina in an apparently resting condition, although
by the cell contents of the vaginal fluid oestrus was shown to
be present. A few animals evidenced continued external signs
of ‘heat’ during the metoestrum and the dioestrous interval, so

THE OESTROUS CYCLE IN THE MOUSE 305

that here again external signs as criteria of a condition of oes-
trus are not reliable. That such signs are due primarily to con-
gestion and consequent poor tonus of the muscle layers of the
vagina is indicated by their rapid disappearance coincident with
the relaxation of the urethral sphincter following death. How-
ever, to settle the question as to their value in diagnosis, the ex-
ternal signs were always noted when taking smears, and as they
were typical in 53.5 per cent of cases, a description is desirable.
Heape’s terminology is followed in describing the phases of the
eycle, with emphasis on the following points: 1) that growth and
congestion continue through oestrus; 2) that the metoestrum
may be further subdivided into two periods.

To eliminate a too frequent use of ‘oestrum’ in its various forms,
the stage will be designated as shown below:

D, dioestrum, period of relative quiescence.

P, prooestrum, period of augmented growth and con-
gestion.

O, oestrum, period of sexual excitement, the climax of
prooestrous conditions.

M, | metoestrum, period of return to the dioestrous

M, J condition.

Stage D. In the mouse during the dioestrous interval the
vulva is very inconspicuous and the orifice of the vagina is usually
tightly closed. There is a little fluid which is viscous and stringy.
The smear shows epithelial cells, usually only a few, in various
stages of nuclear degeneration and cytoplasmic shrinkage, and
always some polymorphonuclear leucocytes.

Stage P. During the prooestrum, if external signs are marked,
the vulva is pink or red and swollen, the vagina gapes open, the
fluid in its lumen is serous, and the smear shows only nucleated
epithelial cells (fig. 2).

Stage O. During the oestrous period, the vulva may still
be swollen, the orifice of the vagina open and a dull white in color,
the vaginal mucosa almost dry, and the smear shows only corni-
fied, non-nucleated, red eosin-staining cell remains (fig. 3). In
animals where external signs are absent or slight, the finding of
only cornified cells in the smear makes a diagnosis of ‘heat’
possible.

306 EDGAR ALLEN

Stage M,. In the first half of the metoestrum, the vulva has
usually lost most of its swelling, the orifice of the vagina still
gapes open and is whitish, occasionally showing small granula-
tions, the lumen is still dry, and the smear shows red cornified
elements of the previous periods, always very numerous, and
now bunched or caked (fig. 4).

Stage M,. This stage is evidenced by a normal vulva and a
tightly or partly closed vaginal opening. ‘The vaginal contents
change as the period progresses from a pasty or viscous to
a fluid consistency, and the smear shows polymorphonuclear
leucocytes among the red horny elements. A few polymorphs
and many cornified cells is an early M, stage, and a heavy leuco-
eytic infiltration and decrease in number of the cornified cells
is a late one (fig. 5).

This stage then merges into D with a decrease in leucocytosis
and the appearance in the smear of small numbers of nucleated
epithelial cells. By standardizing the technique of smear prep-
aration, stages O and M, are easily differentiated, although the
basis for this division is the number and clumping rather than
any change in the character of the cells themselves.

The separate phases as well as the duration of the whole cycle
show great variation under the most uniform conditions of food
and environment. Further discussion may be postponed until
the histological condition of the various genital organs can be
described.

5. ANIMALS STUDIED HISTOLOGICALLY

A series of twenty-seven carefully timed animals was prepared
for histological study. They consisted chiefly of four strains:
albino, black, brown, and gray. Observations were made daily
for a period of from three to five weeks before killing, in order
to obtain an accurate record of the number and duration of oes-
trous periods as an aid to the interpretation of ovarian condi-
tions. The smear made just before killing usually followed
the routine smear for that day by three to six hours, so that
two observations at short intervals were available for diagnosis.

THE OESTROUS CYCLE IN THE MOUSE 307

The criteria used in placing the animals are the following:

1. The cell contents of the vaginal smear at death.

2. The number of layers of vaginal epithelium unaffected by
cornification or leucocytic infiltration.

3. The position, with regard to the free surface of the epithe-
lium, of the granular and horny layers when present.

4, If ova are present in the uterine tubes, the segment in which
they lie indicates the time of ovulation with regard to oestrus.

5. The stage of development of the corpora lutea when present
aids in a correct arrangement of the series. Their age is estimated
by the position of the ova in the tubes, and compared with cor-
pora lutea of pregnancy (recorded in column 7, table 1).

The following table of animals killed for histological exami-
nation represents a complete, closely spaced series. It seems
convenient to begin at the middle of the dioestrous interval and
return to the same point in the next cycle.

The results of the histological examination of this series
of organs can be most satisfactorily presented by means of
a detailed description of typical animals (marked x in table 1)
representing the midpoints of the five stages of the cycle.

The dioestrous interval

Stage D is best represented in this series by animal *2, a
virgin mouse seven months old. She was killed after 53 days,
observation, during which time nine complete cycles were re-
corded, making the average duration 5.9 days. In six of these
nine cycles pronounced external signs were apparent. The last
four oestrous periods occurred 3 to 5, 11, 14 to 15, and 19 to 20
days before her death. The record shows her to have been in
an early day of the dioestrous interval after a cycle of from 7 to
8 days’ duration marked by external signs and containing a three-
day oestrous period.

A gross examination of the internal genital organs was made
immediately after opening the body cavity before there was any

1 These underlying layers are very difficult to count in some of the phases,
so that two numbers are used to express a limit of error.

308 EDGAR ALLEN

TABLE 1

Animals examined histologically arranged in order of progressive phases of the
oestrous cycle as diagnosed by vaginal smears and microscopical exami-
nation of the genital organs. (For key to table, see footnote.)?

& a) j Zz & &
eects: oe Ey ae Be
Pat Be Aa I Pe Gr:
uals ro de pe | aa 524
@2!| 6 | a&s oa ae Bg 2 BE6 0
pale cl Sch esl “tcl Si ues ai : a
Basin ilvetie pale a hey eeeneaa) olaeae a
1 | .26 |, -5-6.| Gone 2nd-3rd | 2+ 30-50 hrs. | ONE, OP | D
#2| 241 56 | Gone 3rd 3 3+ days | INE, 3P | D
3 8| 5-7] Gone 3rd+ 4 3+ days | INE, 2P | D
A |» 1) ), 56 1| Gone None re ONH,1P | D
5 | 13) 3-5 | Gone None 5 ONE, 1P isp
6} 15) 5-6 | Gone None ONE, OP | D
7 6 Gone None ? ONE, OP | D
8 3 | 7-10| Before for- | 3rd 3 0C, INE.) 2
mation
x9 | 10} 12-13) Granular, no} None 34 3+ days | INE, OP | P
herny
10 | 12 | 11-13} Under 24 None 34 3+ days | ONE, OP | P
11 | 14] 11-13] Under 2-4 None ? 0C, INE JP
12 9] g-10| Under 3-5 None 5 0C, 1NE | P
13 | 23] 9-10| Under 3-4 *None | None} * 1C; INE ee
14 1 | 10-11} Superficial None 5 1C O
#15 4 | 98-32) Superficial None 5 1C O
16 2 None 5+ 10 O
17 | 16 | 12-13] Superficial None 7-8 1C O
18 | 21] 9-12) Superficial 1st-2nd | 1 3- 7 hrs. | 3C, Bu M;
#19 | 19 | 11-12] Superficial 2nd 2 30-35 hrs. | 3C, Bu Mi
20 | 18 | 10-12} Superficial *None None| * 2C, Bu Mi
21 7 | 46 | In lumen 2nd-3rd | 2 20-40 hrs. | 2C, Bu Mi
22} 20] 6-7 | In lumen 2nd-3rd | 2 40-50 hrs. | 8C, Bu Mi
23 | 25} 4-7] In lumen 3rd 3 50-60 hrs. | 2C, 1P M2
24 | 17| 7-9 | In lumen 3rd 3 60-70 hrs. | 1C, 1P Me
25 | 22| 7-8] In lumen 3rd 3 50-60 hrs. | 2C, 2P Me
*26 | 27 | 6-8 | In lumen 2nd-3rd | 2 & 3} 30-50 hrs. | 1C, 3P M2
27 5 | 5-6] In lumen 3rd 3 72+ hrs. | 1C, 2P M2

2 Key to table 1. Column 1 represents the progressive order of the series
beginning with an early phase of the dioestrous stage. #, Animals described
in detail as typical of the five stages of the cycle; * (in columns 5 and 7), ovula-
tion not spontaneous; 0, 1, 2, $ (in column 8), represent relative numbers of cell
types; C, cornified epithelial cells; NE, nucleated epithelial cells; P, polymorpho-
nuclear leucocytes.

~

THE OESTROUS CYCLE IN THE MOUSE 309

obliteration through contraction of the characteristics which
differentiate between the O and D uterus. The anastomosing
uterine and ovarian vessels were small, the uterus was of medium
size and anemic.

Histological examination of cross-sections of the vagina mid-
way between the cervix and the vulva shows the lumen to be
small in diameter, with the walls thin, folded, and collapsed.
Free nucleated epithelial cells and leucocytes are present in
considerable numbers. There is no basement membrane under
the epithelium and the deepest layer of the stratum germina-
tivum shows no distinct cell membrane on the side adjacent to
the stroma. The epithelium not extensively infiltrated with
leucocytes is only five or six layers deep and shows no signs of
cornification (fig. 6). Very few mitoses are to be found. Scat-
tered leucocytes are abundant in the one or two superficial layers,
and in several places small clumps are gathered in clear lacunae.

In general the description of the vaginal epithelium holds
true for that of the lower cervical canal; there is no basement
membrane beneath the epithelium, which is five or six layers
high, and no sign of cornification.

Cross-sections through the uterine cornua midway between
their junction and the attachments of the oviducts show narrow,
slit-shaped lumina indicative of a lack of distention. There
are very few cells of any sort free in the lumina. The epithelium
has no basement membrane, the lower parts of its cells stain
lightly, and cell walls are not readily distinguishable. In many
places the cells are piled up four to eight layers deep in poorly
staining syncytial masses. Mitoses are only occasional, being
present in a few instances in the syncytial masses. ‘The epithe-
lium is intact everywhere, although it is quite heavily infiltrated
with leucocytes. Some of the glands show functional activity,
as evidenced by slight distention.

The oviducts were adherent to the periovarian sacs and were
sectioned serially with the ovaries.

The oviducts are moderately distended throughout, and are
entirely free from leucocytic infiltration. The non-ciliated
epithelium, lining the second and third portions, shows few signs

9

310 EDGAR ALLEN

of degeneration, but the ciliated cells covering the highly folded
mucosa of the part leading from the ovisac show all stages of
the extrusion of their nuclei. Some of these extruded nuclei
still adhere to the free surface of the cells and are similar to
nuclei of normal cells. Others show varying degrees of pycnosis.
Many small regions are to be found devoid of cilia, apparently
marking cells from which the nuclei have been extruded. A
further description of this process will be undertaken later.

The two oviducts in this animal contained eight ova, still in
good condition, although in none was the zona pellucida promi-
nent. (This may be due to the fixing reagent as stated by
Sansom, ’20.) In most of the ova the second maturation spin-
dles were still intact, but rarely were polar bodies recognizable.
The ova were bunched in the last segment of the tubes. There-
fore, ovulation occurred three days previously (H. P. Smith,
dais

The ovaries contain eight medium-sized follicles, all superfi-
cially located. The primary liquor folliculi is beginning to form,
but has not yet reached a stage far enough advanced to make
possible the distinction of a cumulus. The nuclei of the ova are
in the resting stage. In this pair of ovaries, there are more than
thirty atretic follicles in all stages of degeneration, most of them
deeply situated in the stroma. There are at least three sets
of corpora lutea present.? The most recent corpora lutea are
easily distinguishable from the older ones by their blue color,
the latter staining more heavily with eosin. There are eight of
this last set. They are similar in degree of development, so
that a description of one will suffice for all. The central lake
is almost obliterated, chiefly by the hypertrophy of the granu-
losa cells which do not at this stage take eosin readily. The
theca interna has entirely disappeared and the ingrowth of
connective tissue has reached the edges of the lake and woven
a fine reticulum about the inner walls of the luteal cells, at the
same time beginning an arrangement of these cells into cords.
Slight vascularization is evident, but there are erythrocytes in

3 It should be understood here that all corpora lutea referred to in this paper
are corpora lutea of oestrus, as no pregnant nor lactating animals have been used.

THE OESTROUS CYCLE IN THE MOUSE 311

the central cavity in only one of the eight corpora lutea of this
set. From the position of the ova in the tubes, it was concluded
that ovulation has occurred three days previous to killing. This
set of corpora lutea of oestrus is, therefore, three days old. They
are surely the equivalent of the seventy-two-hour corpora lutea
of pregnancy.

The next older corpora lutea are greatly degenerated. They
are small, deeply placed, irregular in outline, and poorly demar-
cated from the surrounding stroma. The proportion of connec-
tive-tissue cells to luteal cells is about equal. As stated above,
the cytoplasm of the luteal cells stains red with eosin, but it is a
faded or blotchy red. No distinct large blood vessels are present
as in the first set described. A third set consisting of only a
few still more poorly defined corpora lutea is present. These
are very small, staining a light pink, and are almost completely
obliterated by a connective-tissue ingrowth.

Of the three sets of corpora lutea distinguishable, the age of
the youngest can be placed at three days, with a limit of error
of about twenty hours. From the results of the examination
of other ovaries of this series, the second set is at least ten days
old, and the third still older.

There are a few cells present in these ovaries which could be
defined as interstitial.

The prooestrum

Animal *9 was chosen as typical of the stage P condition.
A routine examination was made at 1 p.m. and another just
before killing at 4.30 P.m., an interval of three and one-half
hours, in which time many of the leucocytes had disappeared
from the vaginal contents, indicating an early stage P. Pre-
vious to killing she had been observed for sixteen days, during
which time four complete cycles had been recorded, making the
average duration four days. External signs were marked dur-
ing all four of the oestrous periods. These occurred 4, 7 to 8,
11 to 12, 15 to 16 days before death. Her record is one of mini-
mum cycle duration and perfect regularity.

312 EDGAR ALLEN

The uterine and ovarian vessels were congested (two or three
times larger than during the D period), and the uterine cornua
were much distended. When the animal was killed the uterus
did not expel this fluid (as the bladder expels the urine) and when
held up between the observer and the light, the uterine cornua
are so transparent that the folds of the mucosa are easily visible.
The ovarian capsules were not distended.

Cross-sections of the vagina show a fairly large lumen con-
taining some nucleated epithelial cells and a very few apparently
degenerate polymorphonuclear leucocytes. The epithelium shows
a basement membrane in a few restricted regions. There are
twelve to thirteen layers of epithelial cells, of which the outer
four to five layers stain very lightly with eosin. This demarca-
tion is made still clearer by a well-formed granular layer which
serves as a line of division. The layers superficial to this are not
so flattened as those immediately underlying the stratum granu-
losum. There are as yet no other signs of cornification (fig. 8).
Mitoses are abundant in the germinativum. The epithelium is
free from leucocytes except for a very few in the most superficial
layer of cells. The nuclei of the stroma are loosely packed and
intercellular spaces are everywhere evident. The sagittal sec-
tion shows the same conditions, except that a thin cornified layer
still remains on the ventral side of the vagina at its opening on
to the vulva.

The cervical epithelium just before it merges into the simple
epithelium of the uterus, although at this point being only from
three to five layers high, is yet divided into two regions, a deep,
darkly staining, and a superficial, lightly staining one. It is
similar in most respects to that of the vagina except as regards
height.

The lumina of the uterine cornua are large. The fluid with
which they are distended is not coagulated by Bouin’s reagent.
There are no cells of any sort free in the lumina. The epithe-
lium is low columnar and has a distinct basement membrane in
all but a few regions. Mitoses are frequent. No leucocytes are
to be found in the epithelium and only an occasional one is in
the subepithelial zone which is most heavily infested by them

THE OESTROUS CYCLE IN THE MOUSE 313

during the D stage. The gland ducts are distended and their
cell outlines are clearly defined. Mitoses are absent, which indi-
cates little growth activity in the glands during hyperfunction.
Glands are distributed evenly throughout the mucosa except
opposite the line of the attachment of the broad ligament.
The nuclei in the stroma are densely packed, possibly because
of distention of the lumen.

The oviducts are slightly distended. Their epithelium is in
good condition in the non-ciliated portions. In the ciliated seg-
ment, all stages in the process of extrusion of nuclei are apparent,
but to a less degree than in the D’stage animal previously de-
scribed. No leucocytes are present.

There are no ova to be found in either oviduct. Therefore,
if ovulation occurred at the last O period, that should have
been at least four days before death.

Sections of the ovaries show a marked hyperemia. ‘There
are two sets of follicles containing liquor, the first one composed
of ten follicles, which are large and distended with considerable
amounts of liquor. They are all superficially located. The
cumuli are intact and still solid masses of cells. The nuclei of the
ova are in a resting condition. The second set of follicles is
less mature, being medium sized, with the liquor still confined
to small pools at the poles of the follicles.

There are several medium-sized atretic follicles in which the
cells of the cumuli have entirely degenerated, leaving the ova
free in the liquor. The granulosa cells also show marked signs
of atresia.

The ovaries of this animal are unusually large, which may be
partly accounted for by their hyperemia, but the unusual num-
ber of corpora lutea is probably the main reason. At first glance
the ovaries appear to be just large masses of corpora lutea.
Three sets are easily distinguishable. Those of the first set are
superficial, medium sized, and stain dark blue. Their central
lakes are completely ingrown, but two of them show small patches
of red blood corpuscles at their centers. They are at least four
days old. :

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 3

314 EDGAR ALLEN

The corpora of the second set are larger than those of the first,
are superficially located, and deep red in staining reaction. The
cells are arranged in ‘cords,’ but their walls are not clear-cut and
the cytoplasm has a blotchy appearance. The edges of these
corpora lutea in section are clearly defined from the surrounding
stroma. Theca interna cells as a distinct layer no longer exist.
This set probably corresponds to the second ovulation before
death, occurring at some time during the O period; therefore,
seven to eight days previously.

A third set of corpora lutea, less clearly defined from the
stroma, red staining, and about the size of the first set, is present.
The stroma has begun to invade their surfaces and the propor-
tion of connective-tissue cells to luteal cells throughout has
increased. ‘The record of this mouse shows an O period eleven
to twelve days before death, so that this third set must be at
least that old. There are a few cells clearly definable as inter-
stitial in these ovaries. They are restricted in distribution to
limited areas near the periphery.

The oestrous period

Animal *15 shows a typical oestrous condition. She had been
examined for twenty-eight days previous to killing, in which
time only three complete cycles had been recorded, making the
average duration 94 days. Her oestrous periods were 6, 16 to
17, and 28 days previous to death. Her last cycle was of five
days’ duration. Her first and second cycles had long dioestrous
intervals, seven to eight days in each case, while the other four
stages required only three to four days.

Upon opening the body cavity, the uterine and ovarian vessels
showed some congestion, though less than that reported during
the prooestrous period. The uterus was moderately distended,
but less transparent. Gross examination shows the same condi-
tions as found in the prooestrum to prevail, although to a less
degree.

The lumen of the vagina in cross-section is much folded and
collapsed, as would be expected from the record of the examina-

THE OESTROUS CYCLE IN THE MOUSE 315

tion before death. It contains a small number of detached
cornified epithelial cells which are very thin in cross-section.
A distinct basement membrane clearly marks off the epithelium
from the stroma. The epithelium is eight to twelve cells deep
under the granular and horny layers, which are now superficially
placed in all regions. A few shrunken blue staining cells with
pycnotic nuclei still remain in the deeper parts of the crypts
between folds in the mucosa. They represent the last stages of
degeneration of the cells of the prooestrous smear. Mitoses
are frequent. There are no leucocytes present in the epithe-
lium (fig. 10).

The cervical epithelium is six to eight layers high, but shows
no cornification as yet. Except for these two points, the de-
Seription of the vaginal epithelium holds good for that of the
cervix; i.e., it has a clear-cut basement membrane, contains
frequent mitoses, and is free from leucocytes.

The lumina of the uterine cornua are moderately distended,
but contain no cells of any sort. The basement membrane of
the epithelium is distinct and heavy and the cells are columnar
(fig. 15)... Mitoses are moderately frequent. No leucocytes are
to be found in the epithelium. Gland lumina show a slight
distention pointing to moderate functional activity, but the
scarcity of mitotic figures indicates little growth. Cell borders
are distinct and the cells show no degenerative changes. AlI-
though a few leucocytes are distributed through the stroma,
only an occasional one is found in the gland epithelium. In
section, the uterus shows slight hyperemia.

The oviducts are moderately distended. In the ciliated
portions nuclear extrusion is still apparent, but to a less degree
than during the P stage. Consequently, larger surfaces present
unbroken ciliation. There are no leucocytes present. No ova
are to be found in the tubes; therefore, if ovulation occurred
at the last oestrus, this must have been at least four days previous
to killing.

There are two sets of normal follicles far enough matured to
show liquor folliculi. The ten follicles of the first set are the
largest found in this series of animals. They are all superficially

316 EDGAR ALLEN

placed, and although the cumuli are still intact, the secondary
liquor folliculi is present in them in small pools. The ova show
resting nuclei centrally placed. The second set of follicles are
medium sized and show only an early beginning of liquor forma-
tion. ‘There are several atretic follicles present in various stages
of granulosa degeneration.

There are from twenty to thirty corpora lutea in this pair of
ovaries, among which at least three sets can be defined. The
most recent ones, ten in number, are superficially placed and
quite large. ‘These corpora stain a light blue in contrast to the
red-staining older sets. They are clearly defined from the
stroma, and their constituent cells which are arranged in cords
show distinct cell walls. Although the ovaries are hyperemic,
there are few erythrocytes to be seen in these corpora lutea, which
is in marked contrast to the small vessels crowded with erythro-
cytes in the four-day corpora of the stage D animal described
above. They must correspond, therefore, to the O period re-
corded on the sixth day before death. The other two sets of
corpora lutea are distinguishable chiefly by size, position in
regard to the surface of the ovary, and proportionate ingrowth
of stroma cells at their surfaces. The cytoplasm of the ‘luteal’
cells of both sets stains a blotchy red. There are a few intersti-
tial cells in these ovaries.

The early metoestrum

Animal ¥*19, representing a typical M, stage, was one of three
females of a litter of homozygous brown mice separated from
males at the time of weaning, and examined daily for the appear-
ance of her first oestrus. An open vagina was first noted on
July 12th, at the age of three and one-half months, the membrane
closing the orifice having ruptured during the night of the 11th.
Routine smears were started on the 13th, and continued for
forty days, during which time five complete cycles were recorded,
making the average duration eight days. If, however, the first
two longer periods are excluded, it brings this average down to
seven days. At only one of these periods did she evidence
external signs of oestrus. Previous ‘heat’ periods are recorded

THE OESTROUS CYCLE IN THE MOUSE She

2 to 3, 9 to 10, 14 to 16, 23 to 24, and 34 days before she was
killed. A gross examination showed the ovarian and uterine
vessels to be relatively small and inconspicuous. The uterine
cornua were small in diameter and quite opaque. The periova-
rian sacs were slightly distended.

Cross-sections of the vagina (fig. 11) show a semicollapsed
lumen crowded with masses of non-nucleated, cornified, deeply
red-staining, epithelial cells. The epithelium has no basement
membrane and the deepest layer of the germinativum is in inti-
mate relation with the connective-tissue cells of the stroma.
There are eleven or twelve layers of epithelial cells under the
granular and horny layers, which are everywhere superficial
and intact except for the delaminated masses in the lumen.
There are very few or no mitoses present in the germinative
layers. Leucocytes are also absent.

The lumina of the uterine cornua are not entirely collapsed,
but contain in several of the sections a few nucleated epithelial
cells. The basement membrane of the uterine epithelium is
entirely lacking and in its place is a broad red-staining band.
The deeper edges of the epithelial cells and the most superficial
stroma cells are involved in it, and their structures contained in
this band become blurred and take the stain poorly. Small
vacuoles are apparent in and among the epithelial cells. A very
few leucocytes have already entered the degenerate band or zone
under the epithelium.

The oviducts are moderately distended. Vacuoles are present
in the non-ciliated epithelium of the segments adjacent to the
uteri and in the ciliated regions extrusion of nuclei is general
(fig. 19).

Eight ova are present in the second segments of the tubes.
Membranes and maturation spindles are intact in every case,
and polar bodies are still adherent. The animal was killed then
during the second day after ovulation.

There are from ten to fourteen medium-sized follicles in which
the liquor folliculi is beginning to form at the poles. There are
none sufficiently distended to indicate the approach of an ovu-
lation. A very few small and medium-sized atretic follicles
are present.

318 EDGAR ALLEN

There are over thirty corpora lutea in these two ovaries. The
most recent set number eight, which corresponds with the num-
ber of ova in the tubes. They are blue staining and have large
central lakes of liquor. Theca interna cells are still evident at
the periphery and connective-tissue sprouts have not yet grown
completely through the granulosa cells. No erythrocytes are
included, indicating that vascularization is not far advanced.
Judging by the position of the ova in the tubes, these corpora
lutea are in their second day of development.

The other corpora lutea are divisible into at least three sets.
They are all red-staining, solid masses of cells, differing chiefly
in size, location, and clearness of demarcation from the sur-
rounding stroma. ‘The second oldest set are much larger than
the recent ones, clearly defined, and peripherally situated. The
third oldest set are smaller than the second, but larger than the
first. They are separated from the germinal epithelium by
several layers of connective-tissue cells. The second and third
sets correspond to the ovulations during the oestrous periods
nine to ten and fourteen to sixteen days previous to death. This
mouse has apparently ovulated spontaneously at every oestrous
period.

The late metoestrum

Animal 26 had been examined for twenty-two successive
days before killing, during which time five complete cycles had been
recorded, giving an average duration of 42 days. External
signs were marked at only two of these five periods, and in one
of these instances they extended into the late metoestrum. Her
previous oestrous periods were 2 to 3, 6 to 7, 11, 16 to 17, and
20 to 21 days previous to her death.

The last smear shows all degrees of epithelial degeneration and
cytolysis. The cornified cells present a varied staining reaction
to eosin, some being bright red, others a faded pink, and a few
almost colorless. Where one cell is isolated in a group of leuco-
cytes its exoplasm is a clear colorless zone, suggesting the
extraction of the eosin staining cytoplasm.

Immediately after killing, gross examination showed hee
uterus to be anemic, small, and opaque.

THE OESTROUS CYCLE IN THE MOUSE 319

The lumen of the vagina is collapsed and the walls much
folded. It is crowded with polymorphs, among which lie big
fragments of delaminated cornified elements. They are very
thin in section. The epithelium is without any vestige of a base-
ment membrane, pointed shoots of the stroma penetrating into
the lower layer of the germinativum. There remain only four
to seven layers of healthy epithelial cells beneath a broad region
which has been reduced almost to the appearance of a fine retic-
ulum by the infiltration of enormous numbers of leucocytes
which lie in its meshes. In regions less affected, small clear
lacunae containing several leucocytes are distributed throughout
the epithelium. No signs of a granular layer exist, and the
cornified layer has been freed from its attachments in most
places. This action is just as marked in the crypts of the mu-
cosa as on the ridges. Leucocytosis is at its maximum in this
animal. In spite of this extended destruction of the epithelium
an occasional mitosis may be found.

The sagittal section shows that the process of leucocytosis
does not extend far through the vaginal orifice, for as it approaches
the vulva the epithelium still appears normal and its granular
and horny layers are superficial but intact. As these layers are
traced back into the lumen, the eleidin granules begin to disappear
and the cells gradually to stain pink and then red until they
merge into a fully formed cornified layer.

The lumen of the uterus is small in diameter and contains an
occasional small mass of free cells. The epithelium in most
regions 1s quite completely degenerated. There is no basement
membrane, but in its place a broad, blurred red or pinkish band
involving the lower part of the epithelial cells and the adjacent
stroma. The epithelium consists for the most part of several
layers of nuclei, which might lead one to classify it _as pseudo-
stratified. But cell outlines are lacking or very indistinct, so
that it represents the appearance of unorganized masses of
nuclei in poorly staining cytoplasm. In a very few restricted
areas occasional mitoses are evident, but for the greater part no
signs of growth activity are apparent. The epithelium and, to
a greater extent, the subepithelial zone are heavily infiltrated
with leucocytes (fig. 16).

320 EDGAR ALLEN

The glands show a minimum of function, as judged by the
slight distention of their ducts. A few mitoses are scattered
through their epithelium. Leucocytes in small numbers are
present between and beneath their cells, but seem to exert little
cytolytic action.

The oviducts, especially the segments adjacent to the uterus,
are distended. ‘The epithelium is high columnar and contains
a few vacuolated cells in the non-ciliated portions, while in the
ciliated section the extrusion of nuclei is marked.

There are seven ova present in the tubes. Some of them
show signs of degeneration. The membranes are lacking and
the chromatin material has disappeared. ‘Two ova in the right
oviduct still show the second maturation spindle and one has a
polar body in which separate chromosomes can be distinguished.
These two ova are in the second segment of the oviducts, while
all the rest from this ovulation are in the third.

Most noticeable in the examination of the left ovary was the
unusually numerous follicles of moderately large size, ranging
from an average diameter of 0.32 mm. to 0.4 mm. There are
twelve of these in the left ovary and seven in the right. The
liquor folliculi is well formed, the cumuli are intact, and the nuclei
of the ova resting. One of these large normal follicles in the
right ovary is sausage shaped and contains two ova, each with
its separate cumulus and its resting nucleus. .

Twenty to thirty corpora lutea are present in both ovaries.
The most recent set are blue staining, but are not all at the same
stage of development. Two in the left, and three in the right
ovary have small central lakes, and two of those in the right
ovary contain erythrocytes plainly indicative of ‘‘bleeding
into the central cavity.’’ The shoots from the cells of the theca
interna have grown completely through the layers of the granu-
losa in these five corpora and have formed five connective-tissue
reticula around the central lakes of liquor. In the other two
recent corpora in the right ovary, the central lakes are much
larger and the connective-tissue sprouts have not yet grown
through the granulosa cells. There is no distinguishable histo-
logical difference in the luteal cells of the two stages. Five of this

THE OESTROUS CYCLE IN THE MOUSE 321

set of corpora lutea are in their third and two in their second day
of development. There are two older sets of red corpora lutea
in these ovaries corresponding to the oestrous periods six to
seven and eleven days previous to killing. The large number
of these in the right ovary as contrasted with the few in the left
would point to a hyperfunction of the former, at least during
the last three oestrous periods. The unusual number of normal,
moderately large follicles in the left ovary indicates the shifting
of the major function for the next oestrus.

There are a few interstitial cells in these ovaries. Leucocytes
do not appear in significant numbers, although leucocytosis is
at its height in the vagina and uterus.

6. SUMMARY OF STAGES BY ORGANS
1. External signs

Although certain external signs may occur during the prooes-
trous and oestrous periods in the mouse, these periods may be
present without them or the signs may continue after ‘heat’
has passed. When present, the external signs consist of a swell-
ing and coloration of the vulva due to congestion and the gaping
open of the vaginal orifice.

2. Vaginal content

Changes in the cells and the fluidity of the vaginal contents are
a much more reliable criterion of the condition of oestrus. Dur-
ing the prooestrum and oestrum there are no leucocytes present
in the vaginal contents. At all other times they are found
there in varying numbers. During the dioestrous interval
(D) epithelial cells (chiefly nucleated) and leucocytes are pres-
ent; in the prooestrum (P) only light staining cells with pycnotic
nuclei; during oestrus (O) single, non-nucleated, eosin-staining
cornified elements constitute the smear; these are clumped in
masses in the early metoestrum (M,), and invaded by leucocytes
as this period progresses until most of the cornified elements dis-
appear and a dioestrous condition again prevails (figs.2 to 5). In
stage D the vaginal content is viscous or stringy; in P it is

aoe EDGAR ALLEN

serous. As the O stage begins the epithelium may become dry,
and then granular in the My period. As the leucocytes invade
the masses of C cells during the M. stage, the contents of the
lumen become pasty, then milky, and finally stringy again as
this stage merges into the dioestrous interval.

3. Histology of the vagina

During the D interval the epithelium is low (three to seven
layers), has no clear-cut basement membrane, and is freely in-
filtrated with polymorphonuclear leucocytes. At the end of this
interval, a basement membrane begins to be evident, mitoses
become more frequent, new layers of cells are added, and leuco-
cytosis ceases. In the P stage growth processes reach a maxi-
mum which thickens the epithelium to ten to thirteen layers.
A distinct basement membrane is present in most places. Then
the outer three to five layers begin to degenerate, as is shown by
their loss of affinity for cytoplasmic stains and the pyenosis of
their nuclei. These two areas become clearly separated by the
formation of a granular layer. This is converted into a cornified
layer (stratum lucidum), which is not superficial as would be
supposed, but underlies three to five layers of nucleated cells.
As the P merges into the O stage, these superficial layers disappear
(probably through autolysis or continued cornification) until
the cornified layer becomes superficial, at which time oestrus is
evident. By this time continued growth has piled up the epithe-
lium to a thickness of twelve or thirteen cell layers under the
stratum granulosum which first formed at about the eighth layer.
The definition of the lower layer of the germinativum from the
adjacent stroma is very clear-cut. As the M, period sets in the
cornified layer begins to be delaminated and the lumen of the
vagina is filled with fragments or masses of C cells. The base-
ment membrane becomes thinner and mesenchymal papillae
indent the lower layers of the epithelium. Leucocytes filter in
from the stroma and collect in the superficial layers of the ger-
minativum. After they have accumulated in considerable num-
bers here, they pass on into the cornified masses in the lumen,
stage Ms, and in a day’s time may completely dissolve them.

THE OESTROUS CYCLE IN THE MOUSE ane

Their continued action in the superficial part of the germina-
tivum reduces these layers to the appearance of a reticulum
containing large clumps of leucocytes. This process gradually
declines until the vaginal epithelium returns to a typical dioes-
trous condition (figs. 6 to 13).

Growth activity seems practically at a standstill in the early
D stage. Toward the end of this interval the growth curve
begins to rise until it attains its maximum in a late stage P, after
which it gradually falls during O and M, to its minimum in the
late M. and early D stages.

4. The uterine changes —

Although the uterus goes through a cycle of changes, they
are far less striking than those occurring in the vagina. Gross
changes consist of a marked hyperemia and distention during
‘the P stage, which diminishes gradually during oestrus and disap-
pears as the M, period progresses. During the D interval the
uterus is anemic.

Histological changes in the uterus consist of periodic growth,
degeneration, and leucocytosis of the epithelial cells. These
phases coincide with those in the vagina. A hyperfunction of
the uterine glands (judged by the distention of their ducts) is the
evident cause of the distention of the uterine cornua during the
P and O stages, at which time they are very transparent. Uterine
glands are distributed everywhere throughout the uterine mucosa
except along the line of attachment of the broad ligament. The
glandular epithelium escapes much of the degeneration and
destruction common to the other epithelial tissues. Mitoses
are most frequent in the glands during the late D and early P
stages, becoming less in number as functional activity increases.
In other words, the growth wave in the glands slightly precedes
that in the uterine and vaginal epithelium.

The shape of the uterine epithelial cells varies chiefly with the
degree of distention of the cornua, and is consequently a poor
criterion of growth. Degenerative processes are first apparent
in this tissue in the fading of the basement membrane (which is
so clear-cut during the P and O stages) into a pink-staining

324 EDGAR ALLEN

band which includes the basal sides of the epithelial cells and the
superficial stroma. This is quite marked in the M, stage before
leucocytosis has begun, which would indicate that the destruc-
tion of the epithelium by leucocytosis is secondary to degenera-
tive changes in that tissue. It is in this subepithelial zone that
the leucocytes collect in greatest numbers and from which they
further invade the epithelium. <A few places appear to be ex-
empt, retaining their healthy appearance. Seldom is a region
found entirely denuded of its epithelium, but this tissue becomes
markedly degenerate (figs. 14 to 16). This material does not
show greater numbers of mitoses in the uterine epithelium near
the openings of the gland ducts than in other regions.

5. Changes in the oviducts

The oviducts apparently escape entirely the periodic leucocy-

tosis, so extreme in the rest of the genital tract. They do exhibit —

definite cyclic changes, however. Earlier in this paper the ovi-
ducts have been divided into segments distinguishable by the
presence or absence of cilia, the height of the folds of the mucosa,
and the thickness of the muscle layers. The segment leading
from the periovarian sac is ciliated, the remaining portion has
simple non-ciliated columnar epithelium.

During the P and O stages the nuclei of the ciliated portion are
ranged in a quite regular row. As the M, phase of the cycle
advances, some of them migrate to the free ends of the cells
which loose their cilia and through which these nuclei are ex-
truded. They may retain the appearance of normal nuclei
or become pycnotic before they are extruded, but when lying
on the free surface of the epithelium, are shrunken and dark
staining. This process reaches its height during the M, and
early D stages, at which time the epithelium may become greatly
vacuolated. It is therefore of degenerative significance (figs.
18 and 19).

The non-ciliated portions of the uterine tubes also show vary-
ing degrees of vacuolization, which seems to result from a hyper-
secretion of this epithelium. As yet it has not been possible to
correlate this with definite phases of the oestrous cycle. |

a

—*

THE OESTROUS CYCLE IN THE MOUSE 325

Ova may be found in the tubes during the early D interval,
and if this is very short, may still be present in the succeeding
P stage. They remain in good condition in the oviduct, with
the second maturation spindles and sometimes the polar bodies
intact, for two and even three days. During the third day, in
the segments proximal to the cornua, they may begin to frag-
ment. Their degeneration here must be by autolysis, as no
leucocytes are present in the lumen of the oviducts. If occa-
sionally they remain intact and pass into the uterine cornua on
the fourth day after ovulation, phagocytosis may be their fate,
for leucocytes are present there if the mouse has not passed the
dioestrous interval.

6G. The ovaries

In a consideration of the ovarian cycle, two main subdivisions
will be made for animals that do and those that do not ovulate
spontaneously (i.e., without the added stimulus of sexual con-
tact). Those only occasionally ovulating spontaneously are
obviously intergrades and will not be considered separately.

Where ovulation is spontaneous, three ovarian structures
are involved: the follicles, the corpora lutea forming after their
rupture, and possibly interstitial tissue. Where ovulation is
not spontaneous there are, of course, no corpora lutea present,
but atretic follicles take on an added significance.

Large, normal follicles are always present in the P and O stages,
while none are to be found in the M, period. This, with the
added evidence deduced from the position of the ova in the tubes,
checked by standardized corpora lutea, shows ovulation to
occur at the end of oestrus. The follicles usually rupture syn-
chronously, but this is not necessarily so. In twenty-seven
animals studied histologically three showed almost a day’s
difference between the position of the ova in the tubes, which is
checked by a difference in the degree of development of the cor-
responding corpora lutea. It takes a period equal to at least
one oestrous cycle for the maturation of the follicle from a medium
sized stage with primary liquor folliculi forming at the poles to a
large one greatly distended with a single lake of liquor (fig. 20).

326 EDGAR ALLEN

Corpora lutea of oestrus in the mouse differ in no details
distinguishable histologically from those of pregnaney during
the first four days of development at least. In only a few cases
is ‘‘bleeding into the central cavity” found. Three days are
required for the hypertrophy of the former granulosa cells and
the ingrowth of the theca interna to completely fill the central
lake of tertiary liquor folliculi. For five or six days (unless
another ovulation intervenes) these newly formed corpora stain
blue with haematoxylin, and are therefore distinguishable until
the next oestrus. After this time they have an affinity for eosin.
If size is taken as a criterion of development, corpora lutea of
oestrus in the mouse do not attain their maximum until an age
of from ten to fourteen days is reached. This usually corre-
sponds with the second ovulation after the one initiating their
growth as corpora lutea. Even at the third ovulation following
their start they may be equal in size to five-day corpora (fig. 24).
Thus, an ovary containing from ten to sixteen large, clearly de-
fined corpora lutea, the result of three ovulations between the
fifth and sixteenth days preceding, may again ovulate. After
twenty days, at which time they are usually not superficially
located, ingrowth of cells from the stroma obliterates their
outlines.

In animals of the first class, then, there are always present in
the ovaries one recent set of blue-staining and two, three, or
four older sets of red-staining corpora lutea, and normal follicles
of medium to large sizes. In the O period follicles attain their
largest size, and the youngest set of corpora are solid and blue
staining. In the M, stage the former corpora stain red and a
more recent set of blue-staining ones are developing in the rup-
tured follicles. At this time (one day after ovulation) they
contain large central lakes of tertiary liquor folliculi (fig. 22).
The largest follicles are medium sized and liquor is forming at
their poles. During the M2 stage the ovary is not subject to
the general leucocytosis occurring in the uterus and vagina at
this time. In the middle of the D interval the blue corpora are
solid and the largest follicles contain one fairly large lake of
primary liquor folliculi. During the P stage the follicles become

THE OESTROUS CYCLE IN THE MOUSE O27

more distended and an increase in size is apparent in the cor-
pora lutea.

In the second class of animals (those which do not ovulate
spontaneously) the follicles attain their largest size during oes-
trus, which may be considerably prolonged. The follicles failing
to rupture, the cumuli disintegrate and atresia begins in the
granulosa cells, continuing until the follicular epithelium is en-
tirely gone. Contained ova may fragment in the late stages of
follicular atresia or they may persist with maturation spindles
intact until most of the granulosa cells have disappeared. The
finding of several distinct sets of abnormal follicles in progressively
later stages of atresia when no corpora lutea are present in the
ovaries, but several oestrous periods have been recorded for the
animal, makes it possible to approximately estimate the time re-
quired for a certain degree of follicular atresia. In late stages
of degeneration, former large follicles are reduced to medium
and even small size. Record of previous oestrous cycles is neces-
sary to properly evaluate the conditions of follicular atresia in
the ovary of the mouse.

Interstitial tissue is present in the ovaries in varying but
usually small amounts in the majority of the animals in this
series. Its distribution is for the most part peripheral. Its
presence or amount is not peculiar to any stage of the cycle.

7. TIME RELATIONS OF THE CYCLE

The duration of the cycle and of its various stages shows
great variability. This is represented in the curve (graph 1)
obtained by plotting the number of cycles against their duration
in days. A total of 563 cycles is included. The mode of the
curve falls at 45 days. An average duration of four to six days
therefore represents the findings.

The dioestrous interval shows a greater variation than any
other stage, lasting from less than a day to as long as fourteen
days. Its length is usually from one to three days, however.
In several cases of an extremely long D interval, the smears may
at times show considerable amounts of cornified cells but the pres-
ence of leucocytes makes evident the diagnosis of the stage D.

328 EDGAR ALLEN

Stage P, as diagnosed by the smear method, may be less
than one day, because leucocytes may not entirely disappear
from the superficial vaginal epithelium until after growth in the
deep layers is well under way. Stage O usually lasts one or two
days, but in several cases unbroken O smears have continued
for nine days, and four days of heat are not uncommon. M,
and M, stages usually last a day each and show little variation.

Curve of cycle length

Number of cycles

ie th ee Ll.
Length of cycles in days

Graph 1

The data accumulated in this study indicate no correlation
between the number of ova produced at an oestrus and the
length of the cycle.

While variability of cycle length is great in the same animal,
it is still greater in different strains. Plotting the number of
cycles against their duration in mice of different coat color gave
the following curves. The mode of the curve for brown coat
color is six days. This strain has the greatest number of unusu-

ee See

THE OESTROUS CYCLE IN THE MOUSE 329

ally long cycles. That for black and albino is four days. AI-
binos in our stock are browns minus the color factor. The mode
for yellow and gray falls at five days. The identity of yellow
and gray is more significant, since these grays are recessive deriva-
tives of the yellow strain. The stock used included a black-
eyed, dominant white mouse which has a lethal factor. Her

Variations in cycle length in different strains

—— Yellow
Fas CUReY,

Number of cycles

Length of cycles in days
Graph 2

average for seven cycles was 7.7 days, which, compared to the
mode of four days for the albino strain, is a striking difference.
This is only one case, but it raises the question of the relation
of this lethal factor to the duration of the oestrous cycle. These
findings indicate that a genetic factor is accountable for some
of the variation in cycle length among different strains of mice.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, No. 3

330 EDGAR ALLEN

As to variation in the same litter, my material is too meager
to warrant any statement. However, the following data con-
cerning four albino mice from the same litter are of interest.
These animals were examined for 146 days, making from twenty-
four to thirty cycles for each animal. The average cycle length
is as follows:

TABLE 2
ANIMALS
53 W 53 WNL 53 WNR 533 E
Average, total... fied... 2. 4.8 5.0 4.1 5.2
Number of unusually long} 0 4 (9, 11, 11, | 1 (8 days) | 2 (0, 10
CYGIOS 5. -2.43 a5 SARC ORE: cree 10 days) days)
Average minus long cycles.....| 4.8 4.8 4.9 4.9

Reference to the first line shows considerable variation, but
if the seven longer cycles, which are approximately twice the
average length, be excluded, there is a difference of only 0.1
of a day, which is well within the limit of error.

It is difficult to generalize on the variation with age of the in-
dividual. The first and second cycles following puberty are
usually longer than the average during later life.

Four mice were kept in a cold room at a temperature varying
from 40° to 55°F. for five weeks during the middle of summer to
see if continued low temperature would retard the cycle. It
resulted only in their building an elaborate, covered ‘house-nest’
in which they spent most of the time. The duration of their
oestrous cycles was not affected.

Tumors appeared in two mice which were undergoing routine
examination, which was continued in one case until the tumor
grew to an inconvenient size. The first cycle after the appear-
ance of the tumor contained a seventeen-day dioestrous interval
succeeded by a one-day oestrus. This was followed by an eight-
day D interval, after which time she was mated. No pregnancy
resulted. In this case sexual activity was at least greatly
retarded.

THE OESTROUS CYCLE IN THE MOUSE 331

8. GENERAL DISCUSSION

The literature on this subject is so extensive that it is practi-
cable to discuss here only that bearing directly on special phases
of this work.

In many particulars the oestrous changes in the mouse are
similar to those described by investigators of other rodents.
Important among the recent workers in this field are Heape
(05), on the rabbit; Konigstein (07), on the rabbit, rat, and
guinea-pig; Rubaschin (’05), Bouin and Ancel (710), L. Loeb
(11), Lams (713), Stockard and Papanicolaou (717), on the guinea-
pig, and Long and Evans (’20), on the rat.

Many of my observations merely establish for the mouse proc-
esses previously described in other rodents. It has been a
pleasure to confirm in many instances the observations of earlier
investigators, but in some respects conditions in the mouse throw
a new light on certain of these sexual problems and a few new
interpretations seem warranted.

In discussing the duration of the cycle, Heape (00) says: ‘‘The
differences in sexual periodicity in polyoestrous mammals in
allied forms and even within the limits of the same species is
due to variation in the quiescent period.’’ In the mouse another
possibility presents itself. The mode of the curve for the cycle
duration in brown mice is six days—an interval of two days
longer than that for black and white and one day longer than
that for yellow and gray. Long ‘heat’ periods are more common
to browns than to the other strains. Therefore, although the
variation in the dioestrous interval accounts for much of the
cycle variation, a good deal of it is also due to the variation in
the duration of the oestrous stage itself. The variation in cycle
length is great sometimes in the same animal and among dif-
ferent individuals from one litter. In the latter, however, it is
less than in different strains of mice.

As stated earlier in the paper, albinos in our stock are browns
minus the color factor. The mode of the curve for whites being
two days shorter than that for browns suggests that with the
loss of the determiner for color a shorter cycle has been effected.
Again the modes of the curves of cycle duration of yellow and

332 EDGAR ALLEN

gray mice coincide, and they are both derived from the same
original pair of animals. These facts point to a genetic factor
as partly responsible for variation in the oestrous cycle and
direct attention to the ovum itself as the ultimate cause.

The present findings of a much shorter oestrous cycle in the
mouse than had previously been suspected explains the apparent
exceptions reported by Kirkham and Smith to an oestrous cycle
of twenty or seventeen and one-half days’ duration.

Kirkham (716) reports a case in which ‘‘a set of normal eggs
in the 2-cell stage was found six days post partem.’” Smith
(17) reports ovulation in a mouse six and one-half days after
parturition. These may be explained by a failure to ovulate
or of the ova to be fertilized at the oestrus following parturition,
and the recurrence of another oestrous period five days later.

1. Oestrous changes in the genital tract

a. The vagina and cervix utert. 1. There is in the mouse
little discharge from the uterus into the vagina such as occurs
in menstruating animals. Uterine epithelial cells are not often
found in the smears. Also, although the uterine cornua during
the P and O stages are greatly distended with fluid, the vaginal
mucosa, especially during oestrus, is usually dry. Apparently
the musculature of the cervix functions as an efficient sphincter.

2. In the mouse the massing of the cornified cells of the vaginal
epithelium in clumps (when a standardized smear technique is
used) indicates that ovulation has already occurred when that is
to be spontaneous. It is therefore of importance in diagnosing
ovarian conditions.

As to the fate of the leucocytes so abundant in the M, stage,
many degenerate in the lumen. It was stated in the descriptive
section that during the D interval the vaginal contents were
viscous and stringy. These stringy masses when spread on
slides show a fine web structure with polymorphs at the inter-
stices entangling varying numbers of epithelial cells. The fine
web processes seem to be made of the greatly attenuated proto-
plasm of the leucocytes.

THE OESTROUS CYCLE IN THE MOUSE 333

3. Long-and Evans (’20) have again called attention to the
formation of the cornified and granular layers of the vaginal
epithelium in the rat and guinea-pig as a remarkable histogenetic
process because they do not form superficially. However, it
should be observed that the overlying layers, although nucleated,
early lose their affinity for stain even before the appearance of
granular layer which precedes cornification (described under
animal 8, fig. 7).

b. The uterus. 1. Gross changes. The attention of investi-
gators has been directed towards the uterus from the very begin-
nings of anatomical study, primarily because of its importance
to the embryo during pregnancy. In the absence of pregnancy
in the primates, its striking changes during the menstrual cycle
have led to its designation as ‘the organ of menstruation.’ In
the mouse the vagina, and also the oviducts to a less extent,
share with it the typical oestrous changes.

2. Destructive histological changes. The amount of epithe-
lial and connective-tissue destruction during the metoestrum
and menstruation has been under continual controversy which
seems to have compromised on ‘‘great variability even within
the same species.’ In menstruating animals extensive removal
of epithelium has been reported and denied, but nearly always
bleeding occurs. Bleeding has also been reported in many of
the lower mammals which periodically exhibit typical oestrus.
Lataste (’87) has recorded bleeding during ‘heat’ in several
European rodents. Stockard and Papanicolaou (717) have
reported an occasional slight bleeding in the guinea-pig. There
is little denudation of the epithelium and only rarely bleeding
during the metoestrum in the mouse. The process is restricted
to the degeneration of the epithelium in situ (it seldom breaks
free from the stroma) and heavy subsequent leucocytosis. This
lack of severe destruction may be accounted for in the mouse
by the rapidly ensuing prooestrum, or period of growth, which
may set in three days after the metoestrum begins.

3. Is leucocytosis primary or secondary? Loeb (11), in dis-
cussing this problem in the guinea-pig, says: ‘“‘It is not very
probable that the changes in the epithelium (of the uterus)

334 EDGAR ALLEN

following ovulation are brought about by a disintegration of
some of the epithelial cells.”” Stockard and Papanicolaou (717),
also working with the guinea-pig, say: ‘“‘Large vacuoles are
to be seen between the epithelial cells, and these are probably
produced by the dissolving power of the leucocytes.’”’ Long and
Evans (’20) state that destruction in the uterine epithelium in
the rat is due to ‘vacuolar’ degeneration. <A closely timed series
of material in the mouse shows distinct degenerative changes
evident in the substitution for the basement membrane of a
light staining zone before leucocytosis begins. It seems more
probable that these vacuoles in the uterine epithelium are com-
parable to those in the oviducts (where no leucocytosis occurs)

and are evidences of degeneration which may be the cause of

the leucocytosis. The evidence from the mouse indicates that
epithelial degeneration is a primary, and the leucocytosis a
secondary, phenomenon.

4. Regenerative changes. Many regions of the surface uterine
epithelium as well as the glands escape destruction during a
single metoestrum in the mouse. Also when active mitosis
begins it does not appear to be restricted to regions adjacent to
the openings of the gland ducts.

5. The distribution of glands. The distribution of glands
throughout the uterine mucosa seems possibly to be of some
significance in the consideration of the implantation of the
blastocysts. Huber (15), in his contribution to the embryology
of the albino rat, calls attention to the even spacing of the im-
plantation sites which are ranged along the sides of the cornua
adjacent to the attachment of the uterine ligaments. In the
mouse, in all the animals studied, glands were distributed every-
where throughout the mucosa except along this line. That
this is the future site of placentae is interesting.

c. Cyclic changes in the oviducts. 1. The oviducts seem to
have been overlooked by most investigators in considering
degenerative changes during the oestrous cycle. So far as I
have been able to find, no cyclic degenerative changes have been
reported in them in the lower mammals. In summarizing the
discussion of the question in man, Novak (’21) disposes of the

fs

THE OESTROUS CYCLE IN THE MOUSE 335

cases of tubal menstruation reported in the literature as for the
most part occurring after hysterectomy from the stump of the
oviduct. These are obviously not normal. Czyzewicz (’08)
(quoted by Novak) reported after the study of six normal ovi-
ducts at different stages of the menstrual cycle that they did not
share menstrual phenomena with the uterus.

The oviducts, uterus, and vagina have a common origin from
the mullerian ducts, so possibly factors causing cyclic changes
in the uterus and vagina may be expressed in some way in the
oviducts. They are not, however, subjected to the periodic
leucocytosis of the rest of the genital tract. The extrusion of
nuclei in the ciliated portion, beginning in the early metoestrum
and continuing in a marked degree sometimes to the middle of
the following prooestrum, may be interpreted as of degenerative
significance, paralleling as it does the stages of ‘‘degeneration
and removal by leucocytosis’”’ in the uterus and vagina.

2. The importance of certain structural features of the ovi-
ducts of the mouse. The careful work of Sobotta (95), Huber
(15), and H. P. Smith (17)-had made the differentiation between
the segments of the oviduct easy. The time of passage of the
ova through the different segments as worked out by Smith,
ovulation being calculated in the light of the observations of
Long and Mark (711), has been used in placing the time of ovu-
lation and consequently the estimation of the age of corpora
lutea.

3. The passage of the ova down the uterine tubes. The
mechanism of the passage of the ova down the greater extent
of the oviducts is still not understood in the mouse. Ciliary
action accounts only for their entry into and passage through
the segment proximal to the ovary. Peristaltic action may
possibly furnish motive power for the rest of their passage. Waves
of peristalsis were not, however, apparent in my material, but
the oviducts were always somewhat distended. Valve-like
folds of the mucosa at the entrance of the tubes into the cornua
have already been mentioned as a possible check to backflow of
fluid from the uterus. The arrangement of the muscle layers
of the oviduct as they merge into those of the uterine cornu is

336 EDGAR ALLEN

identical with that in the ‘bile duct sphincter’ recently emphasized
by Mann (20). It is possible that they serve such a function
here. Surely, a back flow of fluid from a distended uterus would
be fatal to the passage of ova down the tubes, if that be by per-
istalsis.

d. The ovaries. Many interesting ovarian problems have
arisen in the course of this work.

a. The follicles and ovulation. 1. Spontaneous ovulation.
Certain species of animals (the rabbit and ferret are examples)
usually do not ovulate spontaneously at oestrus. There has
been much discussion concerning this question in the mouse.
Tafani (89), Sobotta (95), and several later investigators claim
an ovulation without added sexual stimulation. Garlach (06)
and Bouin and Ancel (’09) state that in the mouse ovulation is
dependent upon coition. That it usually occurs spontaneously
the first day after parturition has been emphasized by many
investigators, and made use of by Long and Mark (’11) in arti-
ficial insemination.

Possibly the size of the ovarian vessels immediately following
parturition may be the deciding factor at this ovulation in some
mice, but it is certain that in many mice not lately pregnant
ovulation is not spontaneous at every oestrus and in some virgin
mice it need not have occurred at all, although several oestrous
cycles have been recorded, which indicates the presence of ripe
follicles. It is obvious, therefore, that the classification of the
mouse as a species ovulating spontaneously is not accurate.
While it is not possible to diagnose the absence of spontaneous
ovulation by actual cell content of the smear, unusually long
oestrous periods in certain animals may indicate a failure or at
least a difficulty in ovulation, for the presence of ripe follicles
seems universal in all animals in the oestrous condition.

2. Is ovulation in litter-bearing animals synchronous? Long
and Mark (’11), in considering the maturation of the ovum of the
mouse, state that ovulation is a synchronous process, although
imperfectly so. Sobotta and Burchard report similarly for the
rat. Huber finds fertilized ova usually bunched in their passage
down the oviduct in the rat, which would indicate a synchronous

prey

THE OESTROUS CYCLE IN THE MOUSE Bot

ovulation. In three mice of this series a distinct difference in
the position of the ova in the tubes and a difference in degree of
development of the corresponding corpora lutea may indicate
that the synchronism is less perfect where mating or artificial
insemination do not occur. In these three mice the ova ovulated
at one oestrus were plainly separated into two groups with a
day’s difference between their ovulation time.

3. The number of ova produced at one ovulation. The num-
ber of ova produced at an ovulation as an indication of the degree
of fecundity of an animal is always of interest. When approached
from the number in new-born litters, the failure of insemination,
faulty implantation, intra-uterine death, and mortality at par-
turition introduce an extensive error. Therefore, the problem
is most accurately solved through ovarian studies.

Counts of recent corpora lutea checked in many cases by com-
parison with the number of corresponding ova in the tubes, the
number of the next older set of red corpora lutea, and also the
number of largest-sized follicles in prooestrous and oestrous
ovaries give a total of 449 ova produced at forty-nine oestrous
periods. This makes an average of nine ova to an oestrus. Of
these 449, 230 were counted in left ovaries and 219 in right, show-
ing the function almost equally divided between the two ovaries
in virgin and non-mated animals. Long and Mark, in a large
series, obtained an average of seven ova from the ovulation
following parturition—a 22 per cent difference. It is possible
that a supervening pregnancy reduces the number of ova pro-
duced at the oestrus following parturition.

4. The possibility of alternation of major function. The
finding of a variation in the numbers of embryos in the horns of
bicornuate uteri has raised the question as to alternation of
major function between the two ovaries. In twenty-one ani-
mals in which data were available for the number of ova pro-
duced at two, three, and sometimes four oestrous periods, four
show marked and two slight alternation of function, while in

4 All large follicles need not rupture, which introduces a slight error into
this data.

338 EDGAR ALLEN

the others the function is quite evenly divided between the two
ovaries.

5. The period required for the growth of follicles from medium
size to ovulation size. My material points to an interval of
surely not more than two cycles (eight to twelve days) and
probably only one (four to six days) between the beginning of
the formation of primary liquor folliculi at the poles of the
medium-sized follicles and the time of their final distention and
rupture (fig. 20).

6. Atretic follicles. An observation which seems almost
universal, although it varies greatly in degree in different ovaries,
is the presence of atretic follicles of all sizes. Some investiga-
tors have assumed that ovulation is always spontaneous and
concluded that follicles atrophy without regard to the stage of
the oestrous cycle. This conclusion does not necessarily follow
the finding of atretic follicles of different sizes in the mouse ovary.
There are two possibilities: first, follicles at any stage of develop-
ment may atrophy or, second, they may attain full size, but fail
to rupture at a certain oestrous period, and begin atrophy and
resorption. The finding of maturation spindles in the ova in
many of these atretic follicles points to their former large and
mature state. Continued atrophy may decrease the size until
there are few granulosa cells present and most of the liquor fol-
liculi has disappeared. It has been possible to trace sets of
atretic follicles corresponding in progressive degree of atresia
to previously recorded oestrous periods through a time equiva-
lent to three full cycles. In some of these the ova have completed
their intra-ovarian maturation stages, in others the nuclei are
in a resting condition. The former points to the follicular
apparatus, the latter to the ovum itself as the cause of atresia,
which seems to be by cytolysis of the follicular epithelium.

b. Corpora lutea. 1. Differences in those of pregnancy and
oestrus. So much importance has been attributed to the cor-
pora lutea that a study of those of oestrus has been followed
with great interest. The time relations of the position of the
ova in the uterine tubes has been so carefully worked out that
an estimation of the age of these corpora is quite accurate.

THE OESTROUS CYCLE IN THE MOUSE 339

Sobotta (95) has standardized the development of the corpora
lutea of pregnancy in the mouse, and carefully timed material
has been available in the Washington University Anatomical
Collection with which to check this work. Therefore, a compari-
son of the corpora lutea of oestrus with those of pregnancy has
been possible. Sobotta states that their persistence and ultimate
size is not altered by conception. Surely, there is no difference
discernible histologically during the first four days, and if their
later size be any criterion as to the degree of their development,
they do not reach a maximum until two new sets have been added,
i.e., elght to twelve days after that ovulation which resulted in
their formation.

2. Do they inhibit ovulation? Beard (’98) has been followed
by many investigators in advancing the idea that corpora lutea
prevent ovulation. The fact that they are present in greatly
hypertrophied form during pregnancy and that ovulation does
not occur at this time seems conclusive enough proof.

Loeb (18) first proved experimentally, by the removal of the
corpora lutea of pregnancy in the guinea-pig after they had
ceased to be essential to the tenure of the fetus, that a new
ovulation could be induced earlier than would have occurred
otherwise. He then extended the work to the corpora lutea of
oestrus in non-pregnant animals and found the same effect of
inhibition on ovulation. Papanicolaou (’20) has confirmed these
experiments. The conclusion drawn was ‘‘the corpus luteum,
itself the result of an ovulation, provides a mechanism prevent-
ing ovulation.’”” However this may be in the guinea-pig, it is
surely not normally applicable to the mouse during oestrus
for (because of the shorter cycle) ovulation occurs when two or
three sets of recent large corpora lutea are present in the ovary.
In litter-bearing animals the corpora lutea constitute no
inconsiderable part of the ovaries. Might not ovulation fol-
lowing their excision be merely an expression of the compensa-
tory hypertrophy of the remaining ovarian tissue? Corpora
lutea of oestrus most surely do not normally prevent ovulation
in the mouse.

340 EDGAR ALLEN

3. Do they exert a destructive influence on the mucosa of the
genital tract? Frankel (’03) attempted to prove that the cor-
pus luteum by means of an internal secretion exerted a destruc-
tive influence on the uterus. Although newly forming corpora
lutea are present during the metoestrum in the ovaries of mice
which ovulate spontaneously, in those which do not, none need
be present. And yet typical metoestrous degenerative changes
in the genital tract occur. Consequently, the corpora lutea can
hardly be considered as the cause of cyclical degeneration.

4, Do the corpora lutea furnish a growth stimulus? Stock-
ard and Papanicolaou think it probable that the corpora lutea
of oestrus exert a protective influence on the uterine and vag-
inal mucosae, because they find well-developed corpora present
during the stages in which no degenerative changes are apparent
in these organs, and because corpora of oestrus begin to retro-
gress before the next metoestrum sets in. They write: ‘The
facts obtained in the present investigation might not fully war-
rant the position that the corpus luteum really exerted an active
protective influence over the uterine mucosa, but they certainly
in no sense suggest, and actually speak against, any injurious
action on the mucosa by the secretion of the corpus luteum.”
This may be taken as a constructive suggestion to combat Fran-
kel’s views. That the corpora lutea are not the principal factors
in the growth phase of the genital tract during the prooestrum
in the mouse is plain for two reasons: 1) In those animals
where spontaneous ovulation is the rule and where two or three
sets of large corpora are always present, these degenerative
changes occur just the same. 2) In animals that never have
ovulated spontaneously, and where consequently no corpora are
present in the ovaries, the same regenerative growth processes
occur. Consequently, Stockard and Papanicolaou’s explana-
tion of growth under the protective influence of a secretion from
the corpora lutea is hardly applicable to the mouse.

Therefore, evidence from the mouse brings strongly into ques-
tion the value of corpora lutea of oestrus, 1) as inhibitors of
ovulation, 2) as sources of destructive hormonal influence, or,
3) of endocrine growth stimulus exerted on the uterus.

THE OESTROUS CYCLE IN THE MOUSE 341

c. The cause of cyclic oestrous changes. Many investigators
have postulated theories from histological evidence. Since 1900
the problems have been approached also by experimental methods
and a complicated mechanism built up on experimental evidence
to account for oestrous changes. A short review of these re-
sults seems advisable.

1. Is the cause of oestrous phenomena inherent in the genital
tract itself? Heape believed that oestrus and menstruation
might occur after the removal of the ovaries. Consequently,
he postulated an extra-ovarian cause of these phenomena. Since
1900, however, accumulated evidence from spayed animals has
proved that without the ovaries the oestrous or menstrual cycle
slowly disappears and the uterus and vagina atrophy. Hal-
ban and others, and recently Stockard and Papanicolaou (’17),
have called attention to a cyclic atrophy following ovariotomy.
This is also evident in the mouse in a periodic appearance at
intervals equivalent to the oestrous cycle of cornified cells in
the smear, always in the presence of leucocytes, however. From
this, cyclic changes inherent in the uterus itself might be implied.
Without the presence of the ovaries, however, no growth or
regenerative processes occur. Might not cyclic degeneration
in the uterus and vagina after spaying be merely secondarily
induced by ovarian influences?

Temporarily let us exclude from consideration as improbable
any inherent cyclic nature of the uterus itself and see if the ova-
ries may supply the cause. Three structures are present there
that might be responsible: the follicles, the corpora lutea formed
after their rupture, and possibly interstitial cells.

2. The follicles or the interstitial tissue. Large follicles
distended with liquor folliculi have almost invariably been re-
ported present during the prooestrous and oestrous periods in
mammals, although the work of Heape (’98) and Leopold (94)
throws some doubt on their being always present in the primates.
Consequently, many investigators believe the ripening follicles
to be the cause of these phenomena. They are present in all
mice studied during the prooestrous and oestrous stages.

342 EDGAR ALLEN

Marshall (’14) believes he has experimentally disproved the
ripening follicles as a factor in the mechanism of cyclic sexual
changes. For criticisms of this work the reader is referred to
Stockard and Papanicolaou (’17) and Robinson (20). It seems
to the writer that Marshall’s conclusions are not justified. Mar-
shall falls back on the ovarian interstitial tissue as the cause of
oestrus. Interstitial tissue in the ovary is a very ill-defined,
intangible substance. Any cells not connective-tissue cells
appearing between the follicles may be called interstitial. If
they bear a resemblance to secreting cells they may be called
typical interstitial cells. They are believed by most investiga-
tors to be epithelial in nature and to have an origin similar to
that of the ova and the follicle cells. The interstitial cells in
the mouse ovary are chiefly peripheral in distribution and not to
be confused with those appearing in many forms in the theca
of atretic follicles or originating from old corpora lutea after
connective-tissue ingrowth. There seems to be no _ periodic
hypertrophy of these cells such as would be expected if they
were the cause of cyclic oestrous changes in the genital tract.

3. The corpora lutea. As has already been pointed out, the
corpora lutea, have had attributed to them the following func-
tions, the inhibition of ovulation even in non-pregnant animals
and the cause of degenerative menstrual changes in the uterus
of primates, and metoestrous changes in the uterus and vagina
of other mammals (by analogy), and, finally, the source of a
protective influence on the vaginal and uterine mucosae.

In mice in which ovulation does not occur spontaneously, and
consequently in whose ovaries there are no corpora lutea, the
normal oestrous cycle goes on regularly. In mice that ovulate
spontaneously several healthy sets of corpora lutea are present
during the whole cycle. There seems to be no escape from the
conclusion that in the oestrous cycle in the mouse (unaffected by
pregnancy) the corpus luteum has no primary causative function.

Since the evidence seems to discredit both interstitial tissue
and corpora lutea as causative factors in the phenomena of the
normal oestrous cycle, the follicles are the only remaining ova-
rian possibility.

THE OESTROUS CYCLE IN THE MOUSE 343

4, Evidence for the follicles. In the mouse and in most mam-
mals studied very large follicles are present in the ovaries during
the prooestrous and oestrous stages and absent during the met-
oestrum and dioestrous interval. The histological evidence
from many sources in all mammals studied excepting certain
primates overwhelmingly points to the presence of large folli-
cles as the cause of oestrous changes. However, no direct ex-
perimental proof has yet been produced.

The cause of long oestrous periods. Animal 20 was diagnosed
by the smear method to be in stage M, when killed. If this
diagnosis was correct, degenerative changes should have begun
in the genital tract and ovulation should have occurred one or
two days previously. On histological examination, however,
the epithelium of the vagina and uterus was found to be in an
actively growing condition and a set of large normal follicles
were present in the ovaries. Four normal oestrous periods
had been recorded for the animal and yet the ovaries contain no
corpora lutea. Consequently, ovulation had not been spontane-
ous and it is probable that the large follicles present at death
would not have ruptured had the animal lived. Her present
oestrus was first apparent four days before her death. Would it
not seem probable, then, that the continued growth phase in the
genital tract might (in the absence of ovulation) be directly
dependent upon the retention of mature ova in large normal
follicles? If so, this is a strong suggestion as to the cause of
oestrus. In mice that do not ovulate spontaneously (therefore
the simplest condition), they are always present during the
pro-oestrous and oestrous stages and absent or atretic during the
met- and dioestrum. As the possibility of the corpora lutea
and the interstitial tissue actively sharing as causes seems slight
in the mouse, all the evidence points to the presence of large
normal follicles as the cause of the growth and congestion of
the anabolic periods and the absence of normal follicles as the
primary cause of the catabolic periods in the genital tract.

In animals that ovulate spontaneously, the rupture of the
follicles is the dividing line between these two phases, while in
animals that require an added stimulus for ovulation, atresia

344 EDGAR ALLEN

of the follicles is coincident with the beginning of the metoes-
trum. This indicates that the growth and congestion of the
pro-oestrous and oestrous stages are caused by maturing ova in
large normal follicles, and the degeneration and removal by
leucocytosis of much of the uterine and vaginal epithelium re-
sults after the extrusion of the ovum from, or its atrophy in the
follicle.

That ovulation is the dividing line and that mice of different
strains have distinct modes of cycle length seems to point to the
maturing ova themselves as the ultimate cause of growth changes
and the absence of them from the follicle as the cause of degenera-
tive phenomena of the oestrous cycle.

9. SUMMARY

1. External signs are unreliable criteria of oestrus in mice.
The presence of cornified cells in the vaginal smear is a much
more accurate indication. When these cells appear in masses,
ovulation has usually occurred.

2. The chief changes in the vaginal epithelium are its rapid
growth, the formation of a stratum corneum, and (after ovula-
tion) its degeneration and removal by leucocytosis. The stra-
tum germinativum is also partly destroyed. It may grow from
four to six to twelve or thirteen layers in thickness in one day.

3. There is considerable degeneration and leucocytosis in
the uterine epithelium which is, however, seldom removed from
the stroma. Bleeding rarely occurs in the mouse, but a heavy
leucocytic infiltration takes place during the metoestrum.

4, Periodic degenerative changes in the oviduct parallel those
in the rest of the genital tract. They are evidenced in extrusion
of nuclei from the ciliated epithelium.

5. Ovulation is the dividing line between the anabolic and
catabolic phases of the oestrous cycle, maturing ova in large
follicles always being present during the pro-oestrum and oestrum,
while newly forming corpora lutea or large atretic follicles re-
place them in the metoestrum.

THE OESTROUS CYCLE IN THE MOUSE 345

6. Ovulation is not always spontaneous in virgin or unmated
mice. In some it is regularly spontaneous, in others sporadi-
cally so, and in a few it seldom occurs without an added stimulus.

7. The average duration of the cycle is four to six days. The
mode of the curve for brown mice is six days; for yellows and
erays, five days; for blacks and albinos, four days. The yellows
and grays were descendants from one pair of mice, the albinos
are browns minus the color factor. Therefore, a genetic fac-
tor seems partly responsible for variation in cycle duration, and
this may be tied up with the determiner for coat color.

8. Ovulation need not necessarily be synchronous in unmated
mice.

9. Pregnancy may reduce the number of ova produced at
the ovulation following parturition.

10. There is no difference discernible histologically between
corpora lutea of oestrus and pregnancy during the first four
days of their development.

11. Corpora lutea of oestrus do not normally inhibit ovula-
tion in the mouse.

12. In mice that ovulate spontaneously two or three sets of
corpora lutea of oestrus may be present at all times; in mice that
do not ovulate spontaneously corpora lutea may be entirely
absent, and yet normal oestrous cycles are experienced in both
types of animals. Therefore, the writer concludes that they
have no primary causative relation to oestrous changes in the
genital tract.

13. As ovulation or the beginning of atresia of follicles is the
dividing line between the anabolic and catabolic phases, and as
the genetic factor summarized in no. 8 points to the ova them-
selves, the conclusion is drawn that the presence of maturing
ova in large follicles is the cause of the prooestrum and oestrus,
and that the removal of the ova at ovulation (or their atresia
if this fails to occur) is the primary cause of the degenerative
changes of the metoestrum.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 3

346 EDGAR ALLEN

LITERATURE CITED

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Bearp, J. 1897 (Quoted from Stockard and Papanicolaou, 1917.) The span
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Bourn, P., ev ANceL, P. 1908 Sur la differenciation d’une membrane propre
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1909 Sur les homologies et la signification des glandes a secretion
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DanteL, J. F. 1910 Observations on the period of gestation in white mice.
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Danrortu, C.H. 1916 Use of early developmental stages in the mouse. Anat.
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FRANKEL, L. 1903 Die Function des Corpus luteum. Arch. fiir Gynaekol.,
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Hawupan, J. 1911 Zur Lehre von der Menstruation—Protective Wirkung der
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Hasan, J., UND Konner, R. 1914 Die Beziehungen zwischen Corpus luteum
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Heart, W. 1900 The sexual season. Quart. Jour. Mic. Sc., vol. 44.
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Hitt, J. P., anp O’Donocuusr, C. H. 1914 The reproductive cycle in the
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Kincery, H. M. 1914 So-called parthenogenesis in the white mouse. Biol.
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1917 Oogenesis in the white mouse. Jour. Morph., vol. 30.

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1910 Ovulation in mammals with special reference to the mouse and
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1916 The germ cell cycle in the mouse. Abstract in Anat. Rec.,
Vole LON ps 217.
1916-17 Prolonged gestation period in suckling mice. Anat. Ree.,
vol. 11.

Kirnkuam, W. B., anp Burr, H. 8. 1913 The breeding habits, maturation of
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LanE-Crayron, J. KE. 1907 On ovogenesis and the formation of the interstitial
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Lataste, F. 1887 Motiere du bouchon vaginal des rouqeurs. Soe. Biol.
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Lors, L. 1911 b The cyclic changes in the ovary of the guinea-pig. Jour.
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1911 ce The cyclic changes in the mammalian ovary. Proc. Am.
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1917 The relation of the ovary fo the uterus and mammary gland
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MACKENZIE AND MarsHatt 1913 Recurrence of heat in certain cases in sows
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PLATES

PLATE 1
EXPLANATION OF FIGURES

2 Epithelial cells of the prooestrous vaginal smear. > 800. Cytoplasm _
takes a blue or purple tinge when stained with haematoxylin and eosin. _

3 Epithelial cells of oestrous vaginal smear. > 800. The nuclei have |
their affinity for basic dye; the cells are cornified and stain a bright red with
eosin. ees

THE OESTROUS CYCLE IN THE MOUSE PLATE 1

EDGAR ALIEN

PLATE 2

EXPLANATION OF FIGURES

4 Stage M; smear. 35. Cells are similar to those in figure 3, but all
degrees of cornification are represented by variable staining affinity, and clumps
or masses of cells are frequent.

5 Alatestage Mzsmear. > 125. Non-nucleated cornified cells of the previ-
ous stage (massed in center) and nucleated epithelial cells of the deeper epi-
thelium surrounded by great numbers of polymorphonuclear leucocytes. Some
of the nucleated cells show clear exoplasmic zones indicating the extraction of
eosin staining cytoplasm without the entrance of the leucocytes into the cells.

302

@.4

THE OESTROUS CYCLE IN THE MOUSE

EDGAR ALLEN

¢

ay 3

* ef
>

te )

*.

.

2

a te

<
&

S
0

gh
mh

PLATES eae ; Be

EXPLANATION OF FIGURES

but chiefly in the superficial layers. There is no clear-cut basement mer
7 Section of half of the vagina of the early P stage. X 60. Two zon

clearly defined by staining reaction before either granular or horny layers ap

(animal no.8). - .

THE OESTROUS CYCLE IN THE MOUSE PLATE 3
EDGAR ALLEN

PLATE 4

EXPLANATION OF FIGURES

8 Vaginal epithelium of a later phase of stage P. X 275. The granular
layer now clearly separates the two zones figured in 7 (animal no. 9).

9 The stratum lucidum of the corneum is forming. > 265. (Animal no. 10.)
Sloughed-off nucleated cells make up the stage P smear. :

10 Section of the vagina in stage O. X45. Corneum is well formed, super-
ficial, and still intact. Free cells form the O smear.

d 356

PLATE 4

SE

THE OESTROUS CYCLE IN THE MOU

EDGAR ALLEN

PLATE 5

EXPLANATION OF FIGURES

11 Vagina in stage My. X 65. Corneum is completely delaminated into
the lumen. Leucocytosis has not yet begun.

12 An early Mz stage of the vaginal epithelium heavily infiltrated with
leucocytes. > 180. Few have as yet entered the masses of cornified cells in
the lumen.

THE OESTROUS CYCLE IN THE MOUSE PLATE 5
EDGAR ALLEN

E

—

a
<o

~

eee TP
hor So

PLATE 6

EXPLANATION OF FIGURES

13 Vaginal epithelium of a late M, stage. > 850. Groups of leucocytes have
dissolved out lacunae in the superficial germinativum and enormous numbers
have invaded the cornified masses in the lumen.

14 Section of the uterine cornu during stage P. > 55. The section does
not show the distention apparent before fixation. Glands are moderately re
distended.

360

6

PLATE

SE

1 IN THE MOU
ALLEN

OESTROUS CYCLE

THE

EDGAR

361

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 3

PLATE 7

EXPLANATION OF FIGURES

15 Mucosa of the uterine cornu during stage O. X 365. Note the clear-
cut basement membrane and high columnar cells.

16 Uterine mucosa during early stage Mz. X 550. The distinct basement
membrane figured in 15 has been replaced by a light pink staining zone con-
taining leucocytes.

362

THE OESTROUS CYCLE IN THE MOUSE PLATE 7

EDGAR ALLEN

PLATE 8
EXPLANATION OF FIGURES

17. Sections of several loops of the oviducts. > 55. Only that in the lower
center is ciliated. Segments are distinguishable by ciliation, degree of folding
of the mucosa, and thickness of the muscle walls.

18 Ciliated epithelium of the late stage P oviduct. 550.

PLATE 8

v

THE OESTROUS CYCLE IN THE MOUSE

EDGAR ALLEN

PLATE 9

EXPLANATION OF FIGURES
19 Ciliated epithelium of the oviduct ina late M stage. > 550. The process

of extrusion of nuclei is quite general.
20 Largest-sized follicle usually found after ovulation, stage M1.
Primary liquor folliculi is restricted to two pools. Granulosa contains many

mitoses. Note interstitial tissue above to the right.

x 170.

5 888.

PLATE 9

ones

367

peat eT

¥ * el

THE OESTROUS CYCLE IN THE MOUSE

EDGAR ALLEN

PLATE 10

EXPLANATION OF FIGURES

21 Large follicle (nearly rupture size) in an early stage of atresia. XX 125.
Several follicles in this set are apparently normal. At right, a medium-sized
follicle containing two ova.

22 Ovary of an early M; stage. 58. In upper right field is newly forming
corpus luteum, not yet redistended. At lower left is one fully redistended. The
age of these corpora is estimated at less than seven hours. _

368 —

10

a
ae pase

~
4

PLATE

~
4

OESTROUS CYCLE IN THE MOUSE
EDGAR ALLEN

=
vy

THE

sy ayTnA |
¢ og bp
ag AST:
BE abe ee

af :

any
oe

369

PLATE 11

EXPLANATION OF FIGURES

23 Ovary and third segment of oviduct of late stage Mz animal. X 501.
In upper left field are three ova bunched in the last segment of the oviduct. Of
the three corpora lutea included in this field, the two at the left surface of the
ovary correspond to the ova in the tubes, the one in the lower right field to the
second oestrus recorded before death. They are easily distinguished by staining
reaction not brought out in the photograph. The ages of these were estimated
at three and nine to ten days, respectively.

24 Two corpcra lutea representing follicles which ruptured at the first and
third Operiods before death. 58. That to left stains blue and shows “‘bleed-
ing into the central cavity.’”’ That to the right has a pink tinge, is ‘corded,’
and deeply placed. Ages are estimated at three and fourteen days, respectively.

O2
SI
oO

PLATE 11

THE OESTROUS CYCLE IN THE MOUSE

EDGAR ALLEN

TE Ty,
Ray

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371

Resumen por el autor, B. M. Patten.
La formaci6n del asa cardiaca del pollo.

Las fases tempranas de! establecimiento del coraz6n del pollo
y los estados ulteriores de division en diferentes cAmaras han
sido cuidadosamente investigados por diversos autores. El
presente trabajo se ocupa de los procesos intermedios algo
familiares, pero hasta el presente menos completamente descri-
tos, de la formaci6n del asa y la diferenciacién regional temprana,
basindose en disecciones, reproducidas en modelos pldsticos,
y en reconstrucciones en cera. Se ocupa de los siguientes
puntos: 1. La formaci6n en el tubo cardiaco de un asa en forma
de U dirigida hacia el lado derecho, y de algunos de los factores
causativos invocados en este proceso. 2. La formacién del
asa cardiaca y la relacién de la torsién y flexion del cuerpo del
embri6n con el proceso de la formacién de dicha asa. 3. La
diferenciacién regional del corazén en el bulbo cardiaco, ven-
triculo, atrio y seno venoso, y los cambios tempranos en cada
una de estas regioncs.

Translation by José F. Nonidez
Cornell Medical Collere, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, APRIL 17

THE FORMATION OF THE CARDIAC LOOP IN THE
CHICK

BRADLEY M. PATTEN

Laboratory of Histology and Embryology, School of Medicine,
Western Reserve University

TWO TEXT FIGURES AND THREE PLATES
INTRODUCTION

Most of the numerous investigations concerning the develop-
ment of the heart in birds have dealt either with the very early
phases of its establishment or with the relatively late steps of
its division into chambers. The intervening process of loop
formation, although it is in a general way familiar to embryolo-
gists, has received much less attention. When I had occasion
to consult the literature for a discussion of the subject, I was
unable to find any connected account with adequate figures.
It has, therefore, seemed worth while to extend and publish
some observations on cardiac-loop formation in the chick which
were originally made in the course of other work.

Records of investigations of the development of the chick
heart appear in some of the earliest works on embryology. The
observations on the heart recorded in such classics as those of
Malpighi (1686), Wolff (1759), von Haller (1767), and Pander
(1817), though all of them are remarkable for their time, are at
present chiefly of historical importance. An interesting summary
of the work of these writers appears in the paper of Lindes (’65).
The early stages in the establishment of the chick heart have
since been dealt with by Afanassiev (’69), Gasser (’77), Duval
(89), His (00), Riikert and Mollier (’06), Graper (’07), Func-
clus (’09), Hahn (’09), Miller and McWhorter (’14), and Reagen
(15, ’17). Since this work has been summarized from the mor-
phological point of view by Lillie (’08), and from the experimental

373

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30. NO 3

374 BRADLEY M. PATTEN

point of view by Reagen (’17), it seems unnecessary to go over
the ground again here. The abundant information available
concerning the establishment of the primordial heart tube places
us in a position to take up without preliminaries the processes
involved in cardiac-loop formation.

The observations recorded here are confined to the period ex-
tending from the establishment of the heart as a nearly straight,
double-walled tube to the period at which the process of loop
formation has been completed and the main regional divisions
of the heart have been definitely established.

The later development of the chick heart, involving the for-
mation of the septa and valves which develop during the division
of the heart into chambers, is dealt with in papers by Lindes
(65), Tonge (’69), Masius (’89), Langer (’94), Griel (’06), and
Hochstetter (06).

MATERIAL AND METHODS

At the outset of the work it became obvious that there was
considerable individual variability as to heart configuration even
among embryos having the same number of somites. The first
consideration was, therefore, the selection of a series of embryos
which would show as nearly as possible the normal sequence of
shape changes undergone by the heart. This phase of the work
was. greatly facilitated by the availability of some 2000 chick
whole-mounts in our laboratory collection. By studying the
heart in a large group of embryos having the same number of
somites, it was not difficult to determine the characteristic heart
configuration for a particular stage of development.

Twelve embryos ranging between 29 and 100 hours of incu-.

bation were selected as showing the characteristic steps in the
formation of the cardiac loop and the early regional differentia-
tion of the heart. Each of the twelve embryos belonging to the
initial series was then carefully matched so that three or four
embryos of each stage, exactly like one another as far as could
be determined, were available for the work. One embryo in
each of these sets was reserved for study as a cleared and stained
entire mount, the remaining embryos were used for dissection
and serial sectioning.

a inet

CARDIAC-LOOP FORMATION IN CHICK 375

Camera-lucida diagrams of the cephalic and cardiac regions
were made from the whole-mount series. In these diagrams
the heart and main afferent and efferent vessels, as far as they
could be made out, were drawn in directly. Later in the work,
the outlines of the heart and main vessels were completed! from
dissections and reconstructions of embryos of corresponding
stages. These diagrams appear as the text figures and serve
to show at the same time the stages worked on and the relations
of the heart to the neighboring structures in the body of the
embryo.

It was found that the configuration of the heart itself could be
worked out very successfully from dissections made in alcohol
under a binocular microscope. In such preparations the heart
shape is beautifully shown by strong reflected light and can be
accurately reproduced with the aid of a camera lucida. Em-
ploying this method, drawings of the same heart were made from
three aspects to the same scale of magnification. By using
dividers to keep the dimensions accurate, it was a relatively
simple matter to make a preliminary clay model of the heart
from the drawings. This model, with its basic dimensions cor-
rect, was then finished directly from dissections of the heart,
which could be rotated and thus studied under the binocular
microscope from all angles.

Although the contours are less likely to be distorted in dis-
sected hearts than in reconstructions, there are certain details
that cannot be made out satisfactorily by the dissection method.
The chief point of difficulty is the region of the sinus venosus in
the older embryos. The manner in which the veins entering
the heart are imbedded in the surrounding structures renders it

1 Although a consideration of the changes in the aortic arches does not come
within the scope of this paper, the condition of the arches at each phase of heart
development here dealt with is indicated in the text figures. For discussion of
the development of the aortic arches, reference may be made to the works of
Boas (’87), Evans (’09), Lillie (’08), and Locy (06).

The cardinal and umbilical veins are indicated in the figures of the later stages
only, because in the earlier stages the position of the embryos is such that these
vessels would have to be superimposed on the heart. Moreover, the early stages

in the formation of the vessels are shown beautifully in the figures of Evans (’09)
and Sabin (’17).

376 BRADLEY M. PATTEN

almost impossible to make clean dissections of this region. In
the older embryos, therefore, the heart and the main afferent
and efferent vessels were reconstructed from serial sections by
the wax-plate method of Born. As far as the principal contours
of the heart are concerned, these reconstructions were found to
conform with the clay models made from dissections. They
furnished, moreover, detailed information concerning the sinus
region and the entering veins, which it had not been found possible
to obtain by means of dissections.

The drawings of the heart shown in the plates were made for
the most part directly from dissections. They contain some
details, however, that were added from the wax-plate reconstruc-
tions. The orientation of the heart in the body of the embryo,
and the relations of the vessels are shown in the text figures,
which are lettered in correspondence with the plates.

THE FORMATION OF THE CARDIAC LOOP

The youngest stage studied is represented by embryos of 9

somites (approximately twenty-nine hours’ incubation), in’

which the heart is a nearly straight tube (fig. 1, A, and pls. 1, 2,
and 3, A). Even when the myo-epicardial folds are first approxi-
mated to each other to form the outer wall of the heart tube,
there is already a tendency for the right lateral margin of the
heart to show a greater convexity than that of the left. This
asymmetry is due, at first, more to unequal dilation of the heart
wall than to actual bending of the entire tube, as is indicated by

the fact that the line of attachment of the dorsal mesocardium .

lies very nearly in the sagittal plane of the body.

The dorsal mesocardium at this stage forms an unbroken sup-
porting membrane throughout the entire length of the heart. In
contrast to the condition in mammals described by Yoshinaga
(21), the ventral mesocardium in the chick is complete, or nearly
so, when it is first formed by the approximation of the two folds
of splanchnic mesoderm which constitute the medial wall of the
cephalic portion of the right and left coelomic chambers. The
ventral mesocardium is, however, a more transitory structure

wae

Hej

CARDIAC-LOOP FORMATION IN CHICK ou

than the dorsal, and even at the 9-somite stage its rupture has
begun in the midcardiac region, although its line of attachment
to the heart can still be discerned (pl. 1, A).

In the chick heart the endocardial tubes are less irregular in
contour and are more completely fused with each other at this
stage than in the mammalian heart of corresponding age. Fur-
thermore, the chick endocardium shows no such early foreshad-
owing of the atrioventricular and the sino-atrial constriction as
has been observed in mammals (Murray, 719; Schulte, 716;
Yoshinaga, ’21). In the later stages studied the endocardium
is of secondary interest, its configuration being determined largely
by the limitations imposed upon it by the myoepicardium. A\l-
though the endocardium has been shown in the figures of our
earliest stages by way of bringing this work into continuity
with that of other investigators, the later changes in its configura-
tion have not been followed in detail.

Between thirty and forty hours of incubation (10 to 18 somites),
there is a marked dilation of the heart, but its most conspicuous
change in shape is due to the bending of the entire middle por-
tion of the heart tube to the right (pls. 1 and 3, AtoE). In this
process of bending, as indeed in the entire series of changes in-
volved in loop formation, there is undoubtedly a considerable
factor of mechanical compulsion. The accompanying graph shows
how much greater the elongation is in the heart tube itself than
is the increase, during the same period of time, in the distance
between the attached cephalic and caudal ends of the heart.
Under such growth conditions, bending of the heart is inevitable.
It is quite logical, furthermore, that this bending should be
lateral because of the impediment offered dorsally by the body
of the embryo and ventrally by the yolk. Why it should take
place to the right rather than to the left is not so clear.

It has been suggested that the bending of the heart to the
right might be due to the entry of a stronger current of blood
from the left omphalomesenteric vein than from the right, the
left vein being conspicuously larger at this stage of development.
Sabin (17) has shown, that while heart contractions begin in
the chick as early as the 10-somite stage, the actual circulation

29 Hours 30 Hours 32 Hours
ay (9 somites) B. (10 somites) C. 2 somites)

D. Ges E 40 Hours FE. 42 Hours

* (20 somites)

44 Hours
(22 somites)

47 Hours
(25 somites)

G. Vel

Fig.1, A toL Camera outl
embryos, showing for each sta

I . ie Liam

ines (X 15) of cephalic and cardiac regions of chick

ge studied the relations of the heart to neighboring
378

J. 65 Hours (33 somites) K. 76 Hours (38 somites)

pie roth
<P

Cuards o

1 MOVE) aie ta 0

Heh saa a \

-

ALV. -7 \
V 0.M.M> 7

Z. 100 Hours (45 somites)

structures in the embryo. These figures are arranged and lettered to correspond
with the detailed drawings of the hearts shown in the plates. J to VJ, aortic
arches I to VI, respectively; A.C.V., anterior cardinal vein; A.J.P., anterior in-
testinal portal; Al.V., allantoic vein; Ao., aorta; At., atrium (d., right), (s., left);
Au.P., auditory pit; Aw.V., auditory vesicle; Bul., bulbus cordis; Cuv. d., duct of
Cuvier; Endc., endocardium; F.G., foregut; Hep.s., stubs of some of the larger
hepatic sinusoids; 7.C.A., internal carotid artery; L.B.W., lateral body wall;
Myc., myoepicardium; Myc*., cut edge of myoepicardium; P.C.V., posterior car-
dinal vein; Sin.-at., sino-artrial region of heart before its definite division; S.V.,
sinus venosus; Vent., ventricle; V.ao.r., ventral aortic roots; V.C., visceral cleft;
V.C.M., omphalomesenteric veins; V.C.M.M., fused omphalomesenteric veins;
V.V., vitelline veins.

379

380 BRADLEY M. PATTEN

of blood is not established until the 16-somite stage. The fact
that circulation does not begin until after the bending of the
heart is well advanced excludes inequality of blood-flow from
consideration as causative factor ontogenetically. There is
still the possibility that the bending of the heart to the right is a
recapitulation of development in some ancestral form where
lateral inequality of the circulation had become established
prior to the bending of the heart, but it would be equally plausible
to urge that the bending of the heart to the right was primary,
and that the left omphalomesenteric vein became secondarily
enlarged owing to the opposition of less resistance to the dis-
charge of its blood.

It has also been suggested that, owing to the direction of the
torsion of the embryo’s body, the heart tube is free to expand to
the right, while it would be obstructed on the left by the swing-
ing of the left side of the body wall toward the yolk. Again we
encounter a disregarded time factor. The heart bending is ini-
tiated before there is any indication of torsion in the embryo.
There is undoubtedly correlation between the two processes in
the sense that the development of the heart would be mechani-
cally impeded, if not stopped, by torsion of the embryo in the
opposite direction. Here also it might be maintained that the
heart bend itself is the primary factor and that the direction of
embryonic torsion follows it as a necessary consequence. Cer-
tain it is that the bending of the embryonic heart to the right is
not peculiar to forms in which torsion is conspicuous. The heart
bend is the more deep seated phylogenetically. It occurs in
the vertebrate stock as far back as the elasmobranchs (Hoch-
stetter, 06) and Dipnoi (Robertson, 713), and is a characteris-
tic feature of heart development in Amphibia (Rabl, ’87). One
would scarcely expect to work out the primary causative factors
of such a long-established process entirely from the ontogeny of
forms as far up the scale as birds.

As has already been stated, the ventral mesocardium has
disappeared by the time the bendicg of the heart becomes ap-
parent. ‘The dorsal mesocardium, which is complete when the
bending process begins, soon ruptures in the midheart region

aes
;

CARDIAC-LOOP FORMATION IN CHICK 381

(pl. 3, C, D), and is rapidly obliterated except at the caudal end
of the heart (pl. 3, E). Thus the heart tube, being attached only
at its two ends, is more free to undergo extensive and rapid
changes in shape and position.

Even before the bending of the heart to the right has reached
its maximum, torsion of the embryo’s body begins to change
the mechanical limitations in the cardiac region. As the ceph-
alic part of the embryo comes to lie on the yolk on its left side
(fig. 1, D, E, F) the heart, no longer closely confined between
the body of the embryo and the yolk, begins to swing somewhat
ventrad and lies less closely against the dorsal body wall of the
embryo (pl. 2; ef. C and D with E and F).

The initiation of torsion has another very definite influence on
the heart. Since torsion involves the cephalic region of the
embryo first and progresses caudad, the body of the embryo
becomes more inclined toward the yolk at the level of the ceph-
alic attachment of the heart than at the level of its caudal
attachment. As a result, the truncus arteriosus is twisted by
the carrying of its attached end away from the yolk before a
similar twisting effect is exerted upon the sino-atrial region of
the heart (fig. 1, E, F, G). This is, I believe, the primary me-
chanical factor in starting the transformation of the U-shaped
bend into the cardiac loop. Once the initial twist is imparted,
loop formation progresses extremely rapidly, for the rate at
which the heart tube is outgrowing the pericardial chamber in
length is exaggerated at this time. ‘The distance between the
attached cephalic and caudal ends of the heart is actually being
shortened by the progress of flexion in the embryo at just the
time when the heart tube is elongating most rapidly (fig. 1,
G, H, I, and fig. 2). The attached truncus and sinus ends of the
heart are thus brought closer together, tightening the loop as it
is formed. In the formation of the loop the truncus and bulbus
swing away from the yolk (i.e., toward the embryo’s right),
and come to lie across the caudal part of the heart, at the etrio-
ventricular constriction (pl. 2, G, H, I). The sino-atrial region
being anchored to the body walls by the remaining part of the
dorsal mesocardium, the ducts of Cuvier, and the omphalomesen-
teric veins, undergoes little change in position.

382 BRADLEY M. PATTEN

It is during this stage that the individual variability previously
alluded to is most conspicuous. The more usual configuration
of the heart is indicated in the figures referred to in the preceding
paragraph where the loop is shown as rather closely twisted.
There were not a few embryos, however, in which the heart stood
out from the body, and was more loosely twisted than in the

Length in Millimeters.

Mee as indicated by oie Hi; en

Fig.2 Graph to show the rate of heart-tube elongation as compared with the
rate of elongation of pericardial region. The heart length was measured along
the original middorsal line of the heart tube from the point of bifurcation of the
aortic roots to the point of convergence of the omphalomesenteric veins. (In the
older embryos the omphalomesenteric veins have begun to fuse with each other.
The caudal point for the measurements had, therefore, to be approximated by
taking it in a given relation to the point of entrance of the ducts of Cuvier.) As
an index of the length of the pericardial cavity, the distance between these same
two points was measured along the middorsal iine of the embryo. All of the
measurements were taken on models made to the same scale of magnification, and
then converted to actual size.

CARDIAC-LOOP FORMATION IN CHICK 383

embryos represented in figure 1, G and H. In the embryos I
have studied this condition seems to be correlated with delayed
flexion rather than with any abnormality of the heart tube. It is
probably to be regarded as within the limits of normal
variability.

In the ventral views of the heart it can be seen that as the
process of loop formation progresses, the extension of the heart
to the right is diminished, and that the loop as it is formed swings
not only ventrad, as has been mentioned above, but also dis-
tinctly toward the sagittal plane (pl. 1, G, H, and I). This
change in position may well be due to the fact that by this stage
the body at the cardiac level has completed its torsion and lies
on its left side, so that the heart is no longer prevented by the
yolk from expanding midventralward.

The heart in the stage when the cardiac loop is first definitely
formed (i.e., in embryos of about 25 somites) has been described
by many investigators as ‘S-shaped.’ Neither the dorsal (pl.
3, H) nor the lateral (pl. 2, H) nor the dextrodorsal view of
the heart as seen in the ordinary whole-mount (fig. 1, H)
can be characterized as ‘S-shaped.’ Only when a heart model
or a heart freed by dissection is rotated, so that a direct ventral
view is obtained, does the ‘S-shaped’ configuration become
recognizable (pl. 1, H).

At the close of the second day of incubation, the cranial flexure
of the embryo is developing extremely rapidly (fig. 1, G, H, I).
As the anterior part of the head is bent caudad, it begins to crowd
the heart loop. As a result the ventricular bend of the loop
moves at first caudad and then dorsad (fig. 1, HtoL). Prior
to the formation of the cardiac loop and its dorsocaudal bending,
the ventricular portion of the heart is cephalic to the atrium, in
the primitive vertebrate position. The bending of the loop
brings the ventricle caudal to the atrium in approximately the
definitive relationship characteristic for adult sauropsida.

384 BRADLEY M. PATTEN

THE REGIONAL DIFFERENTIATION OF THE HEART

Certain text-book diagrams of the chick heart show bulbar,
ventricular, and atrial regions, separated by conspicuous con-
strictions while the heart is still in the straight tubular stage.
Although perhaps suggestions of such constrictions are to be
detected at this early stage of development, I have not been able to
satisfy myself as to their definite appearance until the heart is
well bent to the right, and they do not appear at all conspicuously
until nearly forty hours of incubation (16 to 18 somites). In
the heart of a chick of 20 somites, the bulboventricular constric-
tion, previously but vaguely discernible, has become quite definite
(pl. 1, F). The atrioventricular constriction is also well marked
by this time (pl. 2, F). The sinus venosus exists rather as the
place of confluence of the omphalomesenteric veins with each
other and with the atrium than as a definite division of the heart.
Nevertheless, the sino-atrial boundary may be said to be fore-
shadowed by an increased conspicuousness of the grooves formed
on either side where the omphalomesenteric veins enter the
heart at an obtuse angle to it (pl.3, F). The apparent deepening
of these lateral grooves is, however, due rather to expansion of
the atrium than to any actual constriction in this region. There
is as yet no demarcation between sinus and atrium dorsally, and
no caudal line of demarcation between the sinus and the omphalo-
mesenteric veins.

For convenience in description, the heart at this stage can
best be compared to a U with its upright limbs attached to the
body of the embryo (fig. 1, F). The ventricle, definitely marked
off both cephalically and caudally by constrictions, constitutes
the bend of the U. ‘The bulbotruncus portion of the heart tube
constitutes the cephalic limb of the U, which is attached to the
body by the aortic roots. The sino-atrial region constitutes the
caudal limb of the U, which is attached by the omphalomesen-
teric veins, the ducts of Cuvier, and the remaining part of the
dorsal mesocardium.

The early changes in the bulboventricular portion of the heart
are already so well known that they require but e, brief summary.

A te are

CARDIAC-LOOP FORMATION IN CHICK 385

In the formation of the cardiac loop, the U-shaped ventricular
bend becomes compressed (pl. 1, F, G, H). Coincidently, the
apex of the bend is dilated so that the ventricle loses its U shape
and becomes more saccular. The same process of dilation short-
ens, almost to obliteration the ventricular portion of the limbs
of the U, so that the atrioventricular canal and bulbus cordis
appear to lead off side by side, from a common ventricular sac
(pl. 1, 1). Meanwhile, as has already been described in connec-
tion with the formation of the loop, the entire ventricle has
shifted more toward the sagittal plane (pl. 1, F to J). It has
at the same time been bent caudad and then dorsad (fig. 1, H
to L). In this manner the apex of the ventricle, which was
originally the most right-hand portion of the U-shaped heart
tube, becomes the most caudal part.

The first external manifestation of the impending division of
the ventricle into right and left chambers shows in the oldest
stages here studied. During the fourth day, a slight groove
appears on the ventral surface of the ventricle, which extends
ecaudad from the angle between the bulboventricular constric-
tion and the atrioventricular constriction (pl. 1, K, L). This
groove in later stages extends still farther caudad and marks
externally the position at which the septum interventriculorum
develops.

In the bulbotruncus region the early changes are shown so
definitely by the figures that little can be added by description.
The most interesting phases of the development of the bulbus
occur in stages of development more advanced than those with
which this study is concerned. They have been described in
detail by Lindes (65), Langer (94), Masius (89), Hochstetter
(06), and Lillie (’08).

The early differentiation of the sinus venosus has been less
fully described and calls for more detailed attention. A definite
dorsal line of demarcation between the sinus venosus and the
atrium, can first be made out at the close of the second day of
incubation (chicks of 25 somites). At this time the middorsal
portion of the sino-atrial region of the heart becomes dilated.
This dilation is situated just where the persistent caudal portion

386 BRADLEY M. PATTEN

of the dorsal mesocardium is attached to the heart. On either
side it is marked off by a groove extending from the lateral con-
striction at the point of entrance of the omphalomesenteric vein,
onto the dorsal surface of the heart (pl. 3, H and I, S-A. c.).
The dorsal dilation thus bounded may now be differentiated
definitely from the atrium as the sinus venosus.

The differentiation of the sinus venosus takes place at the
same time as the caudal bending of the cardiac loop. It is pos-
sible that their appearance may be more than casually coincident.
The caudal bending of the loop causes the blood from the omphalo-
mesenteric veins to be directed against the dorsal and ceph-
alic wall of the sino-atrial chamber, rather than toward the
atrioventricular ostium, as in earlier stages of development
(pl. 2, H, I, and J). It will be noted that the sinus dilation
occurs at precisely the point at which the blood current impinges
against the heart wall. A deduction that the blood current is a
causal factor in the dilation is alluring, but in default of experi-
mental evidence, any suggestion to this effect must be considered
as purely tentative.

Whatever molding effect the blood stream may exert in the
process, the demarcation of the sinus venosus becomes more
and more distinct as the caudal bend in the heart becomes more
pronounced (pls. 2 and 3, J to L). The caudal bending of the
loop, too, results in the shifting of the sinus from its original
position, caudal to the atrium, to the dorsal position it occupies
at the end of the fourth day of incubation (pls. 2 and 3). At
this stage the sinus venosus is a pouch-like dilation which receives
the ducts of Cuvier laterally and the fused omphalomesenteric
veins caudally. It is marked off from the atrium by a groove,
which is especially strongly developed caudally and dextrally.
Already it opens into the atrial chamber somewhat to the right
of the midline, foreshadowing its later association with the right
atrium.’

2 At this stage the two layers of splanchnic mesoderm which constitute the
dorsal mesocardium flare out on either side and are reflected over the ducts of
Cuvier at their points of entrance into the sinus venosus (pl.3, I). These trans-
verse folds of the mesocardium have been designated (Lillie) as the mesocardia
lateralia.

CARDIAC-LOOP FORMATION IN CHICK 387

The most conspicuous change in the atrial region is its lateral
expansion. As early as forty hours the future atrial region is
dilated so that its transverse diameter is greater than that of
any other part of the heart tube. From the first the dilation to
the left is more marked (pl. 3, E, F). When the bulbus is thrown
against the right side of the atrium in the formation of the car-
diac loop (pls. 2 and 3, H), it seems to crowd the less developed
right atrium and retard its development still more. After the
configuration of the loop has changed so that the bulbus slips
by the atrium, and crosses the heart at the atrioventricular con-
striction (pls. 2 and 3, I), the right atrium begins to expand more
rapidly, but the size of the two atria does not become equalized
until after the stages here under consideration.

The first indication of the separation of the atrium into two
chambers appears in chicks of 29 to 30 somites (53 to 55 hours).
A longitudinal sulcus develops at this time on the ventrocephalic
face of the atrium. In a ventral view of the heart this sulcus
is at first concealed by the truncus and bulbus; but as it becomes
more clearly marked, its caudal portion can be seen extending
toward the atrioventricular constriction (pl. 1, J. K. L, 7-a.g.).

This interatrial groove is an external manifestation of the
formation of the septum superius. (The septum superius or atri-
orum of the chick heart corresponds to the septum primum of the
mammalian heart. In the chick no septum secundum is formed.)
The formation of the interatrial groove does not appear to be
dependent on pressure exerted on the atrium by the truncus
arteriosus. When the groove first appears, an appreciable space
separates the truncus from the atrium. With further growth,
however, the truncus appears to sink into the cephalic portion
of the interatrial groove, and the auricles expand rapidly on
either side of it. Under these later conditions, the truncus prob-
ably does play a secondary part in the division of the atrium in
the sense that it acts as a constricting band on either side of
which the auricles expand.

388 BRADLEY M. PATTEN

SUMMARY

The early phases in the establishment of the chick heart and
the later stages of its division into chambers have already been
carefully investigated by many workers. This paper covers the
somewhat familiar, but heretofore less completely described,
intermediate processes of loop formation and early regional
differentiation.

The work is based on dissections from which plastic models
were made and on wax-plate reconstructions from serial sections. —
It deals with:

1. The formation in the heart tube of the U-shaped bend to
the right and some of the alleged causative factors in this process.

2. The formation of the cardiac loop and the relation of tor-
sion and flexion of the body of the embryo to loop formation in
the heart. ;

3. The regional differentiation of the heart into bulbus cordis,
ventricle, atrium, and sinus venosus, and the early changes in
each of these regions.

Since these phases of heart development all involve complex
changes in configuration and relations, the figures constitute a
graphic summary much more satisfactory than a written résumé.
The shortness of the intervals between the phases of development
figured allows the continuity of the processes to be followed
readily.

CARDIAC-LOOP FORMATION IN CHICK 389

LITERATURE CITED

AFANASSIEV 1869 Zur embryonalen Entwickelungsgeschichte des MHerzens.
Bull. de l’Acad. imp. des science de St. Pétersbourg, T.13, pp. 321-335.

Boas, J.E.V. 1887. Ueber die Arterienbogen der Wirbelthiere. Morp. Jahrb.,
Bd. 138, S. 115-118.

Born, G. 1889 Beitriige zur Entwickelungsgeschichte des Siugethierherzens.
Archiv. f. mikr. Ant., Bd. 33, S. 284-877, pls. XIX to XXII.

Duvat, M. 1889 Atlas d’embryologie. Masson, Paris.

Evans, H.M. 1909 On the development of the aortae, cardinal and umbilical
veins and other blood-vessels of vertebrate embryos from capillaries.
Anat. Rec. vol. 3, pp. 498-518.

Funccius, T. 1909 Der Cervicothorax der Amnioten. Topogenetische Stu-
Aien. (Under direction of A. Fleischmann.) I. Der Prothorax der
Vogel and Sauger, Morph, Jahrb., Bd. 39, 8. 370445.

Gasser, E. 1887 Ueber Entstehung des Herzens bei Végelembryonen. Arch.
mikr. Anat., Bd. 14, S. 459-469.

GrapPer; L. 1907 Untersuchungen uber die Herzbildung der Végel. Arch. fiir
Entwmnk. der Organismen, Bd. 24, 8. 375-410.

GreiL, A. 1903 Beitrige zur vergleichenden Anatomie und Entwickelungsge-
chichte des Herzens und des Truncus arteriosus der Wirbelthiere.
Morph. Jahrb., Bd. 31, S. 123-310.
Figures and conclusions from Greil’s unpublished work on the chick
heart given by Hochstetter, ’06, and Kerr, 719.

Haun, H. 1909 Experimentelle Studien iiber die Entstehung des Blutes und der
ersten Gefisse beim Hithnchen. Arch. fiir Entwmnk. der Organismen,
Bd. 27, 8S. 337-433.

Hertwic, O. 1906 Handbuch der vergleichenden und experimentellen Ent-
wickelungslehre der Wirbelthiere. Fischer, Jena.

His, W. 1900 Lecithoblast und Angioblast der Wirbelthiere. Abhandl. d.
Math-phys. Klasse der Konig]. Sach. Gesellsch. d. Wissenschaften, Bd.
26, S. 173-328.

HocustetTer, F. 1906 Die Entwickelung des Blutgefisssytems. Hertwig’s
Handbuch, etc., Bd. 3, Teil 2.

Kerr, J.G. 1919 Textbook of Embryology. Vol. II, Vertebrata with the ex-
ception of Mammalia. Macmillan, London and New York.

Lancer, A. 1894 Zur Entwickelungsgeschichte des Bulbus cordis bei Végeln
und Siugetieren. Morph. Jahrb., Bd. 22, 8. 99-112.

Linpes, VonG. 1865 Beitriige zur Entwickelungsgeschichte des Herzens. Dis-
sertation, Dorpat.

Linu, F. R.. 1908 The development of the chick. Second ed., 1919. Holt,
New York.

Locy, W. A. 1906 The fifth and sixth aortic arches in chick embryos with com-
ments on the condition of the same vessels in other vertebrates. Ant.
Anz., Bd. 29, S. 287-300.

Mastvus, J. 1889 Quelques notes sur le développement du coeur chez le poulet.
Arch. Biol., T.9, pp. 403-418.

390 BRADLEY M. PATTEN

Miuuer, ApAM M., anp McWuorter, J. E. 1914 Experiments on the develop-
ment of blood vessels in the area pellucida and embryonic body of the
chick. Anat. Rec., vol. 8, pp. 203-227.

Murray, Henry A., Jr. 1919 The development of the cardiac loop in the rab-
bit, with especial reference to the bulboventricular groove and origin
of the interventricular septum. Am. Jour. Anat., vol. 26, pp. 29-39.

Rast, C. 1887 Ueber die Bildung des Herzens der Amphibien. Morph. Jahr-
buch, Bd. 12, 8. 252-274.

ReaGcen, F. P. 1915 Vascularization phenomena in fragments of embryonic
bodies completely isolated from yolk-sac blastoderm. Anat. Rec.,
vol. 9, pp. 329-341.
1917 Experimental studies on the origin of vascular endothe-
lium and of erythrocytes. Am. Jour. Anat., vol. 21, pp. 39-175.

Ropertson, JANE I. 1913 The development of the heart and vascular system
of Lepidosiren paradoxa. Quart. Jour. Mic. Sci., vol. 59, pp. 53-182.

RitcKert, J., unD MoutuieR, 8. 1906 Die erste Entstehung der Gefiisse und des
Blutes bei Wirbelthieren. Handbuch der Vergl. u. exp. Entw., Hert-
wig Baal dela iweane Vi.

Sapin, F.R. 1917 Origin and development of the primitive vessels of the chick
and of the pig. Carnegie Institution, Contributions to Embryology,
no. 18, vol. 6, pp. 61-124.

Scouts, H.W. 1916 The fusion of the cardiac anlages and the formation of
the cardiac loop in the cat. Am. Jour. Anat., vol. 20, pp. 45-72.

Tonar, M. 1869 On the development of the semilunar valves of the aorta and
pulmonary artery of the chick. Phil. Trans. Roy. Soc., London, vol.
159, Pt. J, pp. 387-411.

Yosutnaca, T. 1921 A contribution to the early development of the heart in
mammalia, with special reference to the guinea-pig. Anat. Rec., vol.
21, pp. 239-308.

EXPLANATION OF PLATES

Series of chick hearts, showing the formation of the cardiac loop and the prog-
ress of regional differentiation. The heart contours were drawn directly from
dissections with the aid of the camera-lucida outlines. In drawing the older
stages, wax-plate reconstructions were used for working out the relations of
afferent and efferent vessels and as a check on the configuration of the heart shown
by the dissections.

In the stages represented in figures E to I torsion has involved the cardiac re-
gion of the embryo. Since torsion affects the more cephalic regions first and prog-
resses caudad, the transverse axis of the body of the embryo is at different in-
clinations to the yolk at the cephalic, and at the caudal end of the heart. The
drawings of the ventral and dorsal views are oriented from the frontal plane, and
those of the dextral views from the sagittal plane of the body, at the level of the
aortic arches. For this reason the sinus region of the heart appears inclined.

The relation of the heart to neighboring structures in the embryo is shown in
the text figures. The lettering of the text figures and plates corresponds
throughout.

ABBREVIATIONS

I-VI, aortic arches I to VI Mes.v., ventral mesocardium
At., atrium (d. right), (s. left) Myc., cut edge of myoepicardium
A-V.c., atrioventricular constriction Myc.F., myoepicardial fusion®
Bul., bulbus cordis S-A.c., sino-atrial constriction
B-V.c., bulboventricular constriction Sin-at., sino-atrial region (before its
Cuv.d., duct of Cuvier definite division)
Endc., endocardium S.V., sinus venosus
Hep.s., stubs of some of the larger he- V.ao.r., ventral aortic roots

patic sinusoids Vent., ventricle
i-a.g., interatrial groove V.O.M., omphalomesenteric veins
z.v.g., interventricular groove V.O.M.M., fused omphalomesenteric
Mes.d., dorsal mesocardium veins

’ During the fourth day there is formed a curious attachment between the
myoepicardium of the ventral wall of the sinus venosus and the myoepicardium
of the ventricle (pl. 2, K, L). I have not seen it described elsewhere. From
work now in progress on later stages of development, I believe this strand be-
comes a broad fusion and serves as a pathway over which one of the main
coronary veins from the ventricle reaches the coronary sinus.

391

PLATE 1

Ventral views (X 25) of chick heart in various stages of loop formation. The
somite number and the approximate incubation age of each embryo are indicated
on the plate.

392

CARDIAC-LOOP FORMATION IN CHICK PLATE 1

BRADLEY M, PATTEN

1-- ~~ Mes. v.

--- V. ao. r.

29 HOURS 30 HOURS 32 HOURS 38 HOURS E 40 HOURS

9 somites 10 somites 12 somites 18 somites

~ Sin-at.

42 HOURS 44 HOURS 47 HOURS
20 somites 22 somites 25 somites

i 53 HOURS

29 somites

J 65 HOURS K 76 HOURS 100 HOURS
33 somites 38 somites 45 somites

393

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 3

PLATE 2

Dextral views (X 25) of same series of hearts shown in plate 1.

CARDIAC-LOOP FORMATION IN CHICK PLATE 2
BRADLEY M. PATTEN

Mes. d.___
Cuv. d_ -¢¥®

yN 29 HOURS 30 HOURS 32 HOURS 38 HOURS 40 HOURS

9 somites 10 somites 12 somites 16 somites 18 somites

20 somites 22 somites

FE 42 HOURS G 44 HOURS 47 HOURS I 53 HOURS
25 somites 29 somites

V O.M.M.

65 HOURS 76 HOURS 100 HOURS
38 somites 45 somites

33 somites

395

PLATE 3
Dorsal views (X 25) of same series of hearts shown in plates 1 and a
a
f ;
! : ! y : : ‘
yy - a . ] , yi : fe
, F J 4 m4 » - 4 9

Cae ; om eee t —— § = ~~? — oa 4
ob | es d :
ie” SET ee pial wa Thies > aa A # : :
: ij ; mae) au 7 a pes fs pio hi ; : ar a oh a =
pen meee Oats 1) se Senne Ae —

Nal oie i wot a j bei
ee ‘ ; ~ Ni a :
: ae ou,

CARDIAC-LOOP FORMATION IN CHICK PLATE 3
BRADLEY M. PATTEN

29 HOURS. 30 HOURS 32 HOURS 38 HOU
RS y
A 9 somites B 10 somites G ie Rene D } y 40 HOURS

16 somites 18 somites

42 HOURS
F G 44 HOURS H 47 HOURS I 53° HOURS
20 somites 22 somites

29 somites

25 somites

Hep. S.

VAO SVE ers

65 HOURS 76 HOURS.
J 33 somites 38 somites 100 HOURS
45 somites

397

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THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, No. 4,
JULY, 1922

Resumen por el autor, R. S. Cunningham.

La reaccién de las células que tapizan la cavidad peritoneal,
incluso el epitelio germinal del ovario, a los
colorantes vitales.

El autor ha observado que las células de la serosa que tapiza
la cavidad peritoneal almacenan los colorantes vitales de una
manera muy caracteristica. Las dos manifestaciones mas sor-
prendentes de esta reaccién consisten en la localizacién de la
concentracioén de los granulos tintéreos en un drea circumscrita
del citoplasma de cada célula y en la formacién de una roseta
perinuclear. Las células mesoteliales de diferentes dreas de la
superficie peritoneal presentan ciertas particularidades en sus
reacciones con los colorantes vitales, las cuales son suficientes
para clasificarlas en grupos mientras que todavia se conforman
con el tipo general caracterfstico. Las variaciones notadas en
las ecélulas de diferentes Areas de la superficie peritoneal consisten
en diferencias en la cantidad de colorante almacenado, las
caracteristicas de la roseta perinuclear y la orientacién de la
acumulacién localizada de particulas tint6reas dentro de la
célula. Las células que cubren al intestino contienen general-
mente la menor cantidad del colorante, mientras que las del
mesotelio esplénico y las del epitelio germinal del ovario con-
tienen la mayor cantidad. El epitelio germinal almacena los
colorantes vitales de una manera especial y caracteristica. Cada
eélula contiene una masa de grdnulos, redonda, oval o en forma
de copa, en la zona infranuclear de la célula; esta masa, en los
ejemplares bien tefiidos, IHlenaba toda la porcién de la célula
situada entre el nucleo y la membrana basal. Por otra parte,
la roseta perinuclear ha sido hallada solo raras veces en las
células del epitelio germinal.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MAY l

THE REACTION OF THE CELLS LINING THE PERI-
TONEAL CAVITY, INCLUDING THE GERMINAL
EPITHELIUM OF THE OVARY, TO VITAL DYES

R. S. CUNNINGHAM
Department of Anatomy, Johns Hopkins University

ONE PLATE (EIGHT FIGURES)

The general trend of the large amount of work carried out on
the nature and significance of vital staining has been to indicate
a definite relationship between all the cells which manifest a
reaction to these dyes in the same way and to the same degree.
That many observers have been extravagant in regard to the
various applications of this theory may be admitted, while the
strict value of the principle is still adhered to. Again it has
been customary to consider as important only those reactions
to this group of dyes in which large, or at least moderate amounts
were stored. All cells having a smaller content were more or
less huddled together and left unstudied. Yet the careful study
of the characteristics in those groups of cells where only minor
amounts of vital dyes are stored may prove of as much interest
and importance as the elaborate study of those cells in which
the power to store these substances is very great. This is, I
think, further suggested in the recent brilliant studies of Evans
and Scott (’20) on the reactions of clasmatocytes and fibroblasts.
This work, though directed towards the differentiation of these
two cell-types in as exact a way as possible, revealed much of
the reaction of the fibroblasts with different exposures to many
vital dyes, and it is practically certain that these observations
will later be of very great value in increasing our knowledge
of the phases of fibroblastic activity.

Since the cells lining the peritoneal cavity do not store vital
dyes in large amounts, they have been among those to which
but little attention has been paid. But the finding that the

399

400 R. S. CUNNINGHAM

reaction of these cells is most specific in character, even though
the amount of dye stored is much less than in the case of the
clasmatocyte, has suggested that this may be a method which
will assist in solving some of the long-discussed questions regard-
ing the relationships which the cells lining different areas of the
peritoneal cavity bear to each other and to other cells.

Goldmann, in his studies on vital staining, gives very brief
consideration to the peritoneal lining cells. In one place (’12,
p. 40) he states specifically that the ‘endothelial serosal lining
cells’ do not store any vital dyes, and in another (’09, p. 45)
he records the observation that the germinal epithelium of the
ovary likewise does not stain in the living animal.

Pappenheim (713) and Pappenheim and Fukushi (’14) agree
with Goldmann that the peritoneal mesothelial cells do not take
vital stains. They use this lack of the ability to store vital dyes
as an argument against the participation of the lining cells in the
formation of the free phagocytic cells of inflammatory exudates.
Tschaschin (713) carried out experiments using collargol, isamine
blue, and trypan blue, and states that he found that the serosal
lining cells remained entirely unstained.

That peritoneal mesothelium stains to some extent, however,
when exposed to vital dyes has been noted by Schlecht, Evans,
Kiyono, and Foot. Kiyono (’14) has given the most elaborate
description of the vital staining of mesothelium, and his are the
only illustrations of vitally stained serosal lining cells which I
have been able to find. His figure (a), plate (1), shows two cells
containing dye from animals which had been vitally stained by
intravenous injections of carmine; these cells he designates as
peritoneal lining cells, but he does not state from which surface
they were obtained. Kiyono also studied the reaction of these
cells to trypan blue in the omentum and found that the distribu-
tion of the blue was precisely the same as that found for car-
mine. He suggests that the reason the trypan blue granules
have not always been found in the mesothelial cells is due to
too long fixation, during which the blue diffuses out of the sur-
face cells. He fixed his omental spread preparations for one
hour and then studied them for the distribution of the dye. He

CELLS LINING THE PERITONEAL CAVITY 401

describes a concentration of the dye-granules near the nucleus,
but suggests that this is due to the greater amount of cytoplasm
in that region, and he intimates that there are as many granules
elsewhere in relation to the amount of cytoplasm. ‘The gran-
ules, which he found, were always small and evenly distributed
without accumulations in any particular areas, or irregular
clumping as has been found in the clasmatocyte. His plate
(1) gives a most interesting comparison between the immense
amount of carmine which has been stored by the clasmatocytes
and monocytes, and the sparse granulation of the serosal cells.
On the other hand, the fibroblasts show amounts of carmine not
very different either in amount or distribution from that of the
mesothelium. Kiyono thinks that his work with vital dyes
indicates a very definite and close relationship between the
serosal cells and the fibroblasts, though he finds no actual transi-
tion between the two cell-types.

Evans (715) has also described the sparse granulation of the
serosal cells, noting that the fine granules of dye were concen-
trated around the nucleus, but his descriptions are very brief
and do not indicate whether or not he agrees with Kiyono in
regard to the more general distribution of dye-granules throughout
the cytoplasm.

Schlecht (’07) also describes the peritoneal mesothelium as
taking vital dyes, but does not differentiate very sharply between
the surface-cells and connective-tissue cells, which probably
also include some true clasmatocytes. His observations are
not very exact with regard to the serosal lining cells, but are
sufficiently so to indicate that there are some vital dye-granules
to be found in the serosal mesothelium.

Tschaschin, on the basis of his-studies with vital dyes (713 a)
and on experimental inflammations (’13 b), has concluded that
the serosal lining cells constitute a specific group, having no
relationship with the connective-tissue elements.

Evans and Scott (20, p. 47) have referred to the subject of
the specificity of mesothelial cells as distinct from fibroblasts
and clasmatocytes in their monograph on the connective-tissue
cells. They agree entirely with Tschaschin in considering the

402 R. S. CUNNINGHAM

lining cells as a distinct strain and state that the use of the azo-
dyestuffs has furnished, in part at least, a means of differentiat-
ing these cells. They do not give any very specific discussion of
the characteristics which the surface-cells display other than
that the granules are always minute and that the spleen manifests
them in the most marked degree. That they have observed
many of the characteristic differences which the cells lining the
peritoneum manifest towards vital dyes is undoubted, and their
opinion is clearly in favor of their specificity, but the analysis
is insufficient for establishing any basis regarding the especial
peculiarities of these cells. Undoubtedly an examination of
these cells with a large number of dyes would yield further
interesting facts. ‘

Ribbert (’04), Schlecht (’07), Goldmann (’09), Kiyono (14),
and Evans (’16a, ’16b) have described the vital staining of
the atretic follicle of the ovary. But none of these authors de-
scribes any staining of the germinal epithelium, Goldmann alone
referring specifically to these cells, stating that they do not store
any vital dye at all.

Foot (19, p. 366) reports the observation that the serosal
cells from the omentum stain deeply with vital dyes, and uses
this reaction to suggest their having a genetic relationship to
free cells formed during inflammatory reactions. In a later
communication (’21, p. 635) he describes the serosal lining cells
of the omentum as containing a few fine granules, and concludes
that most of the dye appears to pass through the cells.

Most of the work that has been done on the cells lining the peri-
toneal cavity has been directed to the solution of the problem of
relationship between these cells and the free cells of inflammatory
exudates. But the serosal lining cells are of interest in other
connections, among these the most important are their relation-
ship to fibroblasts and to the fundamental physiological activities
in which the surface-cells are adapted to mediate. The phenom-
ena of vital staining is of very great importance in differentiat-
ing cell-groups from cell-groups and has already assisted very
much in outlining the absolute separation of serosal cells from
clasmatocytes. Can it also be used to assist in establishing

PGR sere 5,

CELLS LINING THE PERITONEAL CAVITY 403

the relationship which exists between the fibroblast and the
serosal cell? Finally, a careful study of the way in which serosal
lining cells react to vital dyes may throw some additional light
on the interrelationships of different areas of mesothelium and
on the physiology of these cells, both individually and as a living
membrane.

MATERIAL AND METHODS

It is obvious at once to anyone who has studied the cells lining
the peritoneal membrane that sections cut in the ordinary manner
perpendicular to the flat surface of the cells could yield but little
information as to the extent or arrangement of the dye concre-
tions in the entire cell. It is, I think, this fact that has caused
so many observers to conclude that the cells are entirely free
from the dye; while most of those who have described the granu-
lation have studied the cells in the omentum where they can be
observed in their entirety by means of spread preparations.

The observations reported here have been made from a series
of experiments on rabbits. The animals were stained with
Griibler’s trypan blue or with carmine prepared according to
Kiyono’s method. Both dyes were usually administered in-
travenously at intervals of twenty-four or forty-eight hours, the
animals received from six to thirty doses of carmine, from three
to twenty doses of trypan blue, and were usually sacrificed one
to two days after the last injection.

In the study of the cells several methods were used. The
animal was anesthetized and the abdomen opened, cells were
scraped from the surfaces of all the viscera, diaphragm, and
body-wall, and were studied immediately. Smears were also
prepared in a similar way, fixed by heat or alcohol and then stained
in an appropriate manner to obtain good contrasts. Spread
preparations of omentum and mesentery were studied fresh.
Omental spreads were fixed by immersion for one hour in osmic
acid or neutral formalin, washed quickly and stained; others
were treated with weak (4 to 1 per cent) silver nitrate, ex-
posed to sunlight, and then fixed in alcohol. Very thin blocks
of all the organs which border the peritoneal cavity were fixed
in neutral formalin for one hour, and then transferred to 80 per

404. R. S. CUNNINGHAM

cent alcohol, dehydrated, and embedded as rapidly as possible.
Blocks were also fixed in 95 per cent alcohol, it having been found
that the dye did not diffuse out to any appreciable extent from
tissue fixed in strong alcohol. Sections were cut parallel to the
surface and all the surface sections carefully preserved in series.
In this way some slight shrinkage of the tissues occurred as
determined by control preparations, but any loss of dye from
the cells was reduced to a minimum. Finally, very thin mem-
branes were obtained by stripping small bits of the surfaces of
organs while the blocks of tissue were in 80 per cent alcohol;
these were stained and cleared, and while many were entirely
too thick for careful study, others were obtained which were
as thin and easily analyzed as omental spreads. In general
there seems to have been some loss even in these preparations,
as could be determined by the control studies in fresh material,
but the loss was in no way sufficient to prevent cellular orienta-
tion, which is obviously the one difficulty in the interpretation
of the cells removed by scraping.

It is a pleasure to thank Mr. Didusch for the care and accu-
racy with which he made the excellent drawings.

EXPERIMENTAL RESULTS

The cells which line the peritoneal cavity may be divided
into four classes by means of the reactions to vital dyes. These
are all closely related to each other, agreeing in certain partic-
ulars which seem to be very fundamental, while they differ in
minor points which are probably in some way related to the
function of the organs which they cover. The classification
into four groups is not to be considered as absolute, because
variations in one may approach the normal in another, but the
general types are sufficiently distinct to require separation.

1. General serosal mesothelium, including the cells covering:
. intestine.

. body-wall.

liver, pancreas, etc.
. mesentery.
. diaphragm.

Be S57 Ss S

i
G

CELLS LINING THE PERITONEAL CAVITY 405

2. Cells covering the omentum.

3. Cells covering the spleen.

A. Cells covering the ovary, the so-called germinal epithelium.

The first division is a composite one, including all the cells
which have no very sharp differences from each other, and
besides all of these cells agree in general in storing smaller amounts
of the dyes than do those of the spleen and ovary and differ
from the cells of the omentum in certain peculiarities other than
the amount of dye stored. The lining cells of the mesentery
are very much like those of the omentum, but have cer-
tain local differences which are more like the general lining cells
elsewhere, thus placing the cells covering the mesentery midway
between the ordinary serosal lining cells and the cells covering
the omentum. In this way there are all gradations between
the cells covering the intestine which take less vital dye than
any other serosal cells, and the cells covering the spleen and the
ovary, both of which show a characteristic vital staining with
considerable ease. The other variations and what evidence
there is of a parallel physiological relationship will be taken up
later.

The most obvious characteristic which has been observed in
the manner in which the lining cells store vital dyes is the collec-
tion of the dye concretions into an area more or less regular in
shape in a definite portion of the cytoplasm. The distribution
of dye-granules in a perinuclear rosette is also characteristic of
the serosal cells, but requires longer exposure to the dye for its
development and is never so universally present as the specific
separate mass in the cytoplasm. These two specific types of
staining are often found combined, but each and particularly
the clump of granules may appear alone or with many minor
variations, which are in part characteristic of the cellular surface,
in part the result of the kind of dye employed, but to a greater
extent due to the length of time during which the cells have been
exposed to the dyes. Again it has been noted in general that
carmine tends to remain more definitely collected in a small area,
while trypan blue tends much more to form perinuclear rosettes,
usually with, but sometimes without, local concentrations. It

406 R. S. CUNNINGHAM

is quite likely that still other variations than those described
would become apparent if a larger series of dyes could be em-
ployed, and they might prove of value in further subdividing
the serosal cells into smaller groups.

General serosal mesothelrum

In animals acutely stained with trypan blue (1.e., having re-
ceived six to eight doses), the cells covering the intestine, the
body-wall, the liver, the mesentery, and the diaphragm had in
common the distribution of dye-granules in a fine irregular, peri-
nuclear rosette with a more or less evident concentra-
tion at some area in the cytoplasm. There were a few scattered
granules besides, usually just adjacent to the small clump. In
animals’ having had only three to five doses, the perinuclear
rosette was usually represented by an occasional granule and
the small cytoplasmic clump at this earlier stage was present
as a small irregular ring of granules. This ring was usually the
first evidence of dye in the cell and often formed an almost per-
fect ring or oval, usually about one-half or one-third the size
of the nucleus. The most conspicuous individual differences
between the individual surfaces included in this group, as shown
in acute staining with trypan blue, were the greater number of
diffused granules in the diaphragmatic cells, the more sharply
formed ring in the mesentery, the greater tendency to perinu-
clear rosettes in the liver, and the generally sharper localization
and less amount of dye in the body-wall and intestine. These
differences are given more because they indicate the relative
lack of extremely sharp differentiation than because they seem
of great importance within themselves.

In animals in which staining was carried beyond eight to ten
doses of trypan blue, the most important changes were a general
increase in the amount of dye around the nucleus in all the cells
of these surfaces except the diaphragm where there was a more
definite increase in the granules diffusely scattered around the
circumscribed area of earliest staining (fig. 2).

CELLS LINING THE PERITONEAL CAVITY 407

Turning now to the reactions of this group of cells to intrave-
nously administered carmine: in the early stages when the blue
was found in a ring with a few scattered perinuclear granules
the carmine was almost always in a small round or oval patch,
always sharply localized in the cytoplasm. These carmine
granules were almost always very small, uniform in size and
evenly distributed. The regularity with which the early carmine
staining showed this precise arrangement was quite remarkable.
When the experimental animal had received more dye, ten to
fourteen injections, the principal difference noted was the in-
crease in the size of the patches of carmine granules and the
beginning of the perinuclear rosettes. In animals in which
the staining was carried much further—fifteen to thirty doses—
the size of the isolated groups of dye-particles increased still
more, and in many cases there were some granules which had
apparently strayed away from the central fold so that these
areas were no longer so sharply outlined. This distribution of
the dye was particularly characteristic of the cells covering the
diaphragm, the irregularity of the granules almost furnishing
sufficient degree of difference to permit the classification of these
cells in a separate group. These diaphragmatic mesothelial
cells were the first to show perinuclear rosettes, though these
were often most irregular and diffuse. Figure 8 shows two cells
which were obtained from the surface of the diaphragm of a
rabbit which had received twenty-five doses of carmine on alter-
nate days; both the diffuse perinuclear arrangement and the
irregularly scattered cytoplasmic granules are shown, but never-
theless there is quite definite evidence of a greater concentra-
tion of the dye in a definite part of the cell. Only in very rare
instances have cells been found from any of the surfaces of the
general serosa that did not present this wholly characteristic
type of staining.

In animals stained over a considerable length of time, surface-
cells other than those of the diaphragm also contained more
carmine granules, but distributed usually with greater regularity.
In those over the stomach and intestine the additional granules
were usually added to the preexisting small ring or mass, while

408 R. Ss. CUNNINGHAM

a few scattered granules sometimes developed about the nucleus.
In the cells covering the liver particularly and the other surfaces
to some extent, the carmine granules, with increased staining,
often developed into a long, oval belt which sometimes crossed
the nucleus only on one side, and sometimes extended partially
if not wholly around it. So that instead of having the cir-
cumscribed perinuclear rosette which has been described as
characteristic of trypan blue, these cells had a rosette in the
form of a belt running all, or part way, around the nucleus antero-
posteriorly.

Finally, it seems important to emphasize the question of the
orientation of the dye distribution in relation to the nucleus and
the position of the cells in situ. These observations were made
both upon living cells and upon sections, the latter being cut
perpendicular to the surface. In sections the perinuclear rosette
so typical of trypan blue was clearly seen as a patch of granules
at either end of the nucleus, one or the other containing more
blue corresponding to the area of localization. This was often
extended somewhat over the surface of the end of the nucleus.
But the characteristic arrangement was the principal amount
of the blue close to one end of the nucleus. In the early staining
with carmine the small sharply localized area of granules was
usually between the nucleus and the surface of the cell, but
placed laterally to the central axis of the nucleus, so that as
the dye increased in amount with longer exposures the cross-
sections showed red granules in both outer and inner zones of
the cell. This again was the formation of the perinuclear belt,
already described, from the lateral or anterolateral group of
granules. Considering the relationship of the early areas of
carmine staining to the center of the nucleus as distributed along
its long axis, they were found to be usually somewhat nearer one
end, but were often close to the center. As the carmine increased
in amount to the maximum, the granules often covered the en-
tire length of the nucleus, but more often formed the perinuclear
belt.

CELLS LINING THE PERITONEAL CAVITY 409

The omentum

When the omentum from a rabbit that had been vitally stained
with trypan blue was spread on a cover-slip, treated lightly with
silver nitrate and stained with carmine, the large pavement
cells of the serosa were found to be very characteristically stained.
They almost always contained a small ring of blue granules in
one part of the cytoplasm, sometimes this ring was entirely
regular and smooth in contour and again it was broken or oval
shaped. These granules appeared in animals which had re-
ceived only a few doses of blue; when the staining was carried
further, the cytoplasm in the neighborhood of this first ring-like
group of granules began to show a few additional granules, and
finally the perinuclear rosette developed. In an animal in which
the staining was carried out with the usual amount of dye, six
to eight doses, the sharply defined area was very characteristic
and the ring was still present though obscured by the develop-
ment of some diffuse granules adjacent to it. The perinuclear
rosette was present in about one-half to one-third of the cells,
but was usually quite irregular. Perhaps the most interesting
and characteristic finding was that, in the majority of the cells,
the ring and its subsequently attached granules were opposite
the long axis of the oval nucleus (fig. 5). Most of the mesothelial
cells covering the omentum were rather elongated, and with the
patches of dye-granules located in one end they presented a very
characteristic appearance. With longer exposures to blue, the
granules gradually extended throughout the end of the cell and
a few appeared irregularly elsewhere in the cytoplasm, increasing
the sharpness of the perinuclear rosette, and sometimes appearing
over or under the nucleus. Occasionally there were two sharp
areas of localization in a single cell, but this was not common in
the normal cells except in those where there were two nuclei;
in such instances the arrangement was usually of one or two
types. In many cells the dye-granules were arranged about
each nucleus as though the cell might be in process of division,
suggesting that each group of granules eventually would be
entirely characteristic of an individual cell. In others there

410 R. S. CUNNINGHAM

were granules massed between the nuclei, and this mass extended
arms of dye which surrounded the two nuclei in more or less
irregular rosettes. In the one case the arrangement suggests
that the two nuclei are functionally the centers of separate
cells, while in the other the arrangement implies that the cell is
truly binucleate and functionally a single unit. The collection
of granules opposite the long axis of the nucleus was not always
present, a few cells in every preparation having the principal
mass at the side of the oval nucleus, but the proportion was
greatly in favor of the other type. A few granules were often
seen scattered between the nucleus and the surface or in the
infranuclear zone, but the principal mass was very seldom in
either of these two regions.

In animals stained with carmine the same easier was
observed with certain modifications. The mass of carmine in
the end of the cell often extended until it almost filled the entire
cytoplasmic area, but always the borders were more regular than
with the trypan blue, the irregular scattered granules being much
less numerous. The perinuclear rosettes were more infrequent,
or rather were relatively later in appearing in the case of carmine.
However, in very heavily stained animals they were to be seen
in many cells, long fine arms of granules extending irregularly
across or around the nucleus, or perhaps out into the free areas
of cytoplasm. A similar condition is shown in figure 7, which
is from the spleen. It is interesting to recall that the cells of
the omentum show much more diffusely scattered granules after
they have been irritated than do the cells of the other areas,
especially of the spleen (Cunningham, ’22), but the significance
of this still remains to be studied.

The spleen

In the cells covering the spleen the perinuclear rosette de-
veloped very early in the course of staining with trypan blue,
but it was usually accompanied by some condensation into the
type of arrangement which has already been described for the
general mesothelium. As has been noted, this blue rosette

CELLS LINING THE PERITONEAL CAVITY All

surrounded the nucleus parallel to its flattened surface, so that
the condensation did not, as a rule, develop between the flattened
surface of the nucleus and the surface of the cytoplasm, but rather
it was adjacent to some point of the periphery of the nucleus.
The numerous apparently different arrangements are all easily
comprehensible if this fundamental type of the distribution of
the dye is clearly understood. Despite the fact that in surface
cells of the spleen the rosettes developed early and quickly,
they contained only a small amount of dye before the condensa-
tion of granules became the most obvious part of the staining.
These collections of dye very rapidly increased in amount until
in smear preparations the cells often seemed to contain only
this single large mass of dye. The location of the mass of dye
in relation to the nucleus was very interesting, and while every
conceivable appearance was found both in smears and sections,
careful study revealed that the dye-masses were, in the vast
majority of cells, located lateral to the flat surface of the nucleus
and near the termination of its shortest diameter in the per-
iphery. The rapid increase in the mass produced the type of
cap seen in figure 4, this cell having been selected because the
perinuclear rosette was represented by only a few granules,
while the entire lateral surface of the nucleus was covered by
the dye. In animals having received about eight to ten injec-
tions of trypan blue, this type of arrangement was very common,
though more granules were usually present in the perinuclear
rosette; the cells shown in figures 3 and 4 were both from an
animal in this stage of staining. In sections from the spleen
at this stage the nuclei of the mesothelial cells were bordered
at either end, the ‘ends’ being obviously the optical appearance—
rather than the anteroposterior poles—of the long axis of the
nucleus, by groups of blue granules varying in amount from a
small number to a large patch, practically filling the cytoplasm
adjacent to the nucleus. The amount of the dye in such sections
was obviously dependent upon where the section divided the
cell in relation to the principal mass of dye.

On further increasing the amount of dye administered, the
edges of the principal dye-mass extended irregularly further and

412 R. S. CUNNINGHAM

further around the nucleus, until, in many cases, in sections,
granules of dye were found in either or both of the supranuclear
and infranuclear zones of the cells. With such increased amounts
of dye administered, the cell-body increased considerably in
thickness usually throughout the entire length, though a supranu-
clear bulge was occasionally present. This arrangement was
constantly found in most of the cells, but there was a small
proportion in which certain characteristic variations appeared.
In some cells the location of the primary concentration was be-
tween the center of the nucleus and the pole of the long axis; in
such cells the extension of the mass led to considerable irregu-
larities in the relation between the nucleus and its cap of blue.
Some of these caps extended around or partly around the pole
of the nucleus in an irregular fashion. Again, a few cells were
noted in which two localized aggregations were situated on oppo-
site sides connected by arms of blue extending around the nucleus
from one to the other. These double caps were found on the
two poles as well as on the two flattened sides of the nucleus,
although they were more numerous in the latter location. Again,
it was found that the lateral mass had extended almost entirely
into the infranuclear zone and had produced a reaction somewhat
similar to that seen in the germinal epithelium of the ovary.
Finally, it must be noted that the lateral location of the dye-
mass, extending a fine outline of granules around the nucleus to
form the typical perinuclear rosette, may, I think, be considered
as the one most striking characteristic of the splenic mesothelial
cells of the rabbit when vitally stained with trypan blue.
Turning now to the results obtained with the intravenous
administration of carmine, the cells of the splenic mesothelium
were found to be equally as characteristic as when stained with
trypan blue. In the early stages the carmine was found dis-
tributed in the usual sharply localized area just lateral to the
center of the nucleus and always superficial to it. As the amount
of dye was increased, the area extended more and more on all
sides, sometimes in a regular fashion, sometimes with fine lines
and a few detached granules; figures 6 and 7 illustrate two stages
in this progressive increase. ‘The perinuclear rosette was some-

——— —

CELLS LINING THE PERITONEAL CAVITY 413

times found at about this time, but was always most irregular
and never so sharp and characteristic as seen with trypan blue.
The area of the cell just over the nucleus, which contained the lo-
calized mass of dye, was humped up often to a considerable
extent. When the staining was carried to the maximum used
in these experiments, the edges of the supranuclear mass extended
irregularly around the nucleus and reached the infranuclear
zone, in this way forming a perinuclear belt, similar to, but much
more extensive than, those found in the cells of the general
serosal mesothelium. The cells had by this stage increased
considerably in size and over a larger surface than at the stage
when there was only a supranuclear swelling. In some few
cells the mass of dye seemed to be around the pole of the nu-
cleus, probably the first-formed area was somewhat displaced
from the region of the center of the cell. This cap formation
around the longitudinal pole of the nucleus was not as common
as with trypan blue. The principal difference observed in the
reactions of the cells to the two types of dyes was the greater
tendency of the carmine to enter the supranuclear zone and to
remain circumscribed, while the trypan blue extended towards
the periphery and usually towards the infranuclear zone. In
general the cells covering the spleen contained considerably
more dye than any other serosal cells at the same general stage
of staining.

It has been reported elsewhere (Cunningham, ’21, ’22) that,
in cats in which the splenic mesothelial cells had been irritated,
the trypan blue granules tended to collect in the infranuclear
zone of the cell together with many highly refractive droplets
which were developed during the progress of the irritation.
Further, it was found that in experimental hydremia in cats
the fluid always collected in the infranuclear zone. The normal
vital staining in the cat likewise tends towards the perinuclear
rosette, but with a considerably greater tendency to the infranu-
clear extension. Whether these differences are very important
cannot be stated as yet, sufficient data on long-continued irrita-
tions of the serosa of heavily stained rabbits not being com-
pleted. The area which shows the most active vital staining

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 4

414 R. S. CUNNINGHAM

probably is important in relation to other activities, or poten-
tial activities, of these cells. No further conclusions can legit-
imately be drawn from these observations at the present time.

The ovary

The cells covering the ovarian ligaments in the rabbit are
the ordinary, normal, flat mesothelial elements, but at the point
of attachment these cells pass by rather an abrupt transition
into the cuboidal cells which constitute the investiture of the
ovary proper. These cells, commonly known as the germinal
epithelium of the ovary, are cuboidal in shape and have clearly
defined, sharply staining nuclei. They seem to rest upon a
very definite basement membrane and the nuclei are placed in
general about midway between the surface and the base.

When vitally stained these cells stored the dye in the infranu-
clear zone, but only in relatively small amounts in comparison
with the amounts which were taken up at the same time by the
clasmatocytes and other cells commonly known as the more
active in their reaction to the vital dyes; so that in lightly stained
rabbits there were very few granules to be seen in these cells,
not more than were found in the cells of the peritoneum generally.
An increase in the amount of stain given caused a greater increase
in the amount of dye which was stored by the cells covering the
ovary, and a very prolonged course of staining produced an
astonishing picture; the amount of dye in the cells increased
very greatly until the bases of the whole layer seemed entirely
filled with the dye (fig. 1). A more accurate description of the
vitally stained germinal epithelial cells is given from one experi-
ment that demonstrates especially well the very large amount
of dye that can be stored: the cells of the germinal epithelium
varied considerably in size and shape, usually they were cuboidal
with a tendency to columnar form, the outer border occasion-
ally tended to be rounded and to show slight indentations be-
tween the cells. The nuclei were relatively clear and stained
well. They were located near the center of the cell, but there
was aslightly wider zone between the nucleus and the basement

CELLS LINING THE PERITONEAL CAVITY 415

membrane than between the nucleus and the surface. Here and
there a cell contained a vacuole in the base of the cell, though
it was very difficult to determine whether these were inter- or
intracellular. Most of the cells had no vacuoles. But every
cell, without exception, contained a group of trypan blue granules
near the basal pole of the nucleus and often forming a slight cup
around it. Careful examination showed that there were a con-
siderable number of types of arrangement; some cells contained
an irregular circle of dye-granules; others a solid, round clump;
others, a cap on the lower pole of the nucleus, and some, an
irregular patch with knobs, projections, etc. Here and there
an occasional granule appeared in the upper part of the cell,
or a line of them extended up around the nucleus in an arm-like
manner. Some of the cells were very heavily laden with granules
which extended often up around half of the nucleus. Practi-
eally all the blue granules were the same size, about $4 to > u
in diameter. None of them was very large, as in clasmatocytes,
and yet few were very fine, as in ordinary normal mesothelial
cells. Here and there one found cells which were considerably
flatter, being about half cuboidal, and here the dye-granules were
again located in the region of the cell just medial to the nucleus.
These flattened cells seemed to be from the edge of the ovary
near its attachment and suggested a transition to normal peri-
toneal mesothelium. When the transition from the flat meso-
thelium to the cuboidal germinal epithelium was studied it was
found to be most interesting. Here transition cells showed less
and less staining in the characteristic area down to the ordinary
mesothelial cell with its patch of granules and perinuclear rosette.

Animals stained with carmine presented precisely similar
pictures, the variations from the reaction to trypan blue were
very considerably less than in areas such as the spleen or dia-
phragm. But there was slight tendency to surround the nu-
cleus, and the irregularities of staining were always more sharply
shown in the animals stained in trypan blue than in those stained
in carmine. In many areas in the sections of the ovaries from
animals stained with carmine, cells were found just beneath the
layer of the germinal epithelium proper and forming a nest-like

416 R. S. CUNNINGHAM

mass bordered beneath by the basement membrane. These
cells were stained exactly like the cells of the surface, having a
small patch of the red granules around one pole of the nucleus,
though they were notoriented in any especial manner. Whether
these cells are derivatives of the layer of germinal epithelium or
whether they are other cells reacting in a similar manner to the
dyes is not yet clear.

DISCUSSION

The characteristic reaction to vital dyes which has been de-
scribed for the general peritoneal mesothelium certainly suggests
that there is a similarity in function which extends throughout
the membrane, and perhaps may even include the germinal
epithelium of the ovary. The differences noted between diverse
areas such as spleen and diaphragm may eventually prove to
be specifically bound up in the peculiar functions of the organs
which they cover, or may be due to differences in environment,
such as blood supply, movements of viscera, or other physical
factors. ‘The characteristic location of the vital dye-granules
in the various types of mesothelial cells may have some bearing
on the differences in the physiological activities of the organs
which the cells cover, but our information is far too limited at
present to even hazard a guess concerning these interpretations.

Obviously, the observations reported here may assist in the
settlement of two general questions. In the first place, there
has been much discussion regarding the relationship between
the fibroblasts and serosal lining cells, some authors having
maintained them to be the same fundamental type of cell mani-
festing different physical characteristics under different environ-
ments, and others having considered them as entirely different
final types. The second question, in regard to which these
findings may be of importance, is the relationship between
the germinal epithelium and the general serosal mesothelium,
on the one hand, and between the germinal epithelium and the
structures of the ovary proper, on the other.

Evans and Scott (’20) have succeeded in separating the fibro-
blasts from the clasmatocytes by their reactions to vital dyes,

CELLS LINING THE PERITONEAL CAVITY A

but they have done even more in giving us a very large number
of figures of fibroblasts stained with various dyes. In studying
their plates it becomes evident that there is no pattern which
one can consider as in common between even a small number of
the fibroblasts.

The typical fibroblast therefore shows no characteristic stain-
ing in a definite area of the cytoplasm such as is so evident in
the serosal cells from the entire peritoneal membrane. Indeed,
the reactions of the fibroblast to vital dyes would seem from
these plates alone to ally them more closely to the clasmatocytes
than to the serosal cells. Many observers have described reac-
tions of connective-tissue cells and serosal cells as being quite
similar during the different stages of inflammations, and on such
grounds as these have considered that they are interchangeable
(Cornil, ’97; Ranvier, ’93; Schott, 09; Weidenreich, ’07; Roloff,
94, 96, and Dominici, ’01, ’02).

Clarke (16), who introduced celloidin and paraffin into the
general connective tissues, considered that he had produced a
true experimental mesothelium which, according to him, was
entirely comparable to the lining cells of the peritoneum. Among
the older workers many thought that the mesothelial cells were
capable of transformation into a ‘variety of forms,’ one of which
was the fibroblast. Schott (09) and Weidenreich (’07) suggest
that the surface-cells of the omentum are merely flattened fibro-
blasts. Mallory (20), in studying tumors of the arachnoid, has
concluded that they are of connective-tissue character, and
because of these tumors arising from the arachnoidal cells,
he concluded that these cells must be considered as flattened
fibroblasts.

The work done on the differentiation of fibroblasts into serosal
cells seems to me inconclusive. That the normal serosal
lining cell has a different reaction to vital dyes from that mani-
fested by the fibroblast is undoubted, but no effort has yet been
made to determine whether the cells which have formed around
foreign bodies or those which have recovered denuded areas of
peritoneum give reactions to vital dyes similar to those reported
here for serosal cells. It seems, therefore, that the evidence so

418 R. S. CUNNINGHAM

far obtained tends to indicate that the serosal cell is a specific
cell-type and is not a transformed fibroblast, in the sense of being
interchangeable morphologically and physiologically.

The relation which the germinal epithelium bears to the ovary
and to the cells lining the general peritoneal cavity has been
extensively studied. In the course of these investigations there
have developed two principal questions, both of which must be
considered in the light of the reactions which these groups of
cells manifest towards vital dyes. In the first place, are the
germinal epithelial cells closely related to the cells lining the
general peritoneum, or are these two groups entirely different
genetically and functionally? In the second place, to what
extent do the follicular cells, interstitial cells, and definitive
ova have their origin in these cells which constitute the ovarian
envelope?

Waldeyer (’70), after long and careful study of many species,
decided that the cells of the general peritoneal mesothelium were
in early stages entirely similar to, and continuous with, the ger-
minal epithelial cells. But later in the developmental history,
all of these cells except those over certain specific areas, differing
in different species, were destroyed or desquamated and their
place taken by subjacent connective-tissue cells. He found that
in the amphibia the cells of the general serosa retained their
primitive characters in wider distribution than in the mammals,
and so he considered that the peritoneal sac might be thought
of as fundamentally a reproductive pouch which gradually was
narrowed until, in mammals, this property was retained only
by the cells of the ovarian envelope.

Gatenby’s work (’16) on the frog, in which he found that the
general peritoneal lining cells could bud off and produce ova, is
in support of Waldeyer’s idea. But, on the other hand, many
observers have considered the cells covering the genital ridge as
being differentiated out of the general coelomic layers.

Neumann (’75) found that there were interesting interrela-
tionships between the cells covering the ovary and those lining
the general peritoneal cavity in the frog. He considered them
genetically identical, because they both arose from the low

CELLS LINING THE PERITONEAL CAVITY 419

eylindrical layer of cells which lines the peritoneal cavity during
its formation from the coelomic layers. Later, the special
characteristics of the germinal epithelium are developed in
connection with their further differentiation into sexual elements.
Furthermore, he found that the flat serosal cells of other areas
in the peritoneal cavity of frogs may be transformed into typical
germinal epithelium when sexual maturity is reached.

MacLeod (80) suggests that the peritoneal epithelium also
undergoes modifications in other regions, e.g., spleenand pancreas,
and Kolossow (’93), in his careful studies of the normal peritoneal
epithelium in both mammals and amphibia, comes to the con-
clusion that the germinal epithelium of the ovary is a part of
the true layer of peritoneal lining cells, which, owing to specific
stimuli, has undergone a certain amount of differentiation. He
also found certain differences in the cells covering the spleen in
mammals and in those covering the stomach of amphibia.

Coert (98) believes that the germinal epithelium is formed
from the general coelomic epithelium and denies its specificity
in Waldeyer’s sense, but agrees with him that the germinal
epithelium gives rise to primordial germ cells.

Van Beneden (’80), Allen (’04), Sainmont (’06), working with
the cat, Firket (14), (20 a), using the chick, and others have
come to the conclusion that the germinal epithelium of the ovary
is a part of the peritoneal mesothelium which has become
cylindrical. Even Waldeyer in his more recent work has modified
his earlier opinions indorsing the conclusions of Coert. In
general all modern work has tended to establish the genetic
continuity of the general peritoneal serosa and the germinal
epithelium; whatever the factors may be that determine the
specific changes which have been found in the cellular layers,
particularly of those over the ovaries, but also to some extent
of those covering the spleen, stomach, etc., they must be asso-
ciated with some stimulus coming from the structures over which
the changes take place.

The results of the vital staining of the germinal epithelium
and the cells of the general peritoneal lining indicate a certain
similarity in the reactions of these two groups of cells. There

420 R. S. CUNNINGHAM

are gradations between the intestine, body-wall, diaphragm,
omentum, and the spleen; the latter approaching the type of
staining manifested by the ovary more closely than the others.
These cells have in common the localization of the dye in a par-
ticular part of the cytoplasm of the cell, and while there is no
direct evidence that similarity in the storage of vital dyes repre-
sents similarity in physiological function, yet it is permissible to
consider such findings suggestive. The extension of such studies
to species such as the frog would be helpful because there is
evidence suggestive of a greater similarity between the general

serosal lining cells and the germinal epithelium in these species.

With regard to the second question concerning the relationship
between the germinal epithelium and the internal structures of the
ovary, a very large amount of work has been done. Elaborate re-
views of the literature have been given by Firket, Coert, von
Winiwarter, and others, and it is entirely unnecessary to repeat
these here. In brief, it has been accepted by most workers that
the medullary cords are downgrowths of the germinal epithelium,
and many also believe the so-called cords of the second prolifera-
tion are likewise derived from the germinal epithelium. The
question about which most of the discussion has been centered
is the relation which the germinal epithelium bears to the defin-
itive ova.

Waldeyer (’70) found large numbers of cells in the germinal
epithelium which he interpreted as young germ-cells, and con-
cluded that these cells were developed entirely from the cells
of the ovarian envelope. This view has been supported in gen-
eral by many workers, among whom are von Winiwarter (’01),
von Winiwarter and Sainmont (’09), Sainmont (06), Lane-
Claypon (’06), Gatenby (16), and von Berenberg Gossler (712).
In this way a general school has been developed whose principal
belief is the origin of the definitive ova either directly from the
germinal epithelium or from the cellular cords which have been
developed as downgrowths from this superficial layer of cells.

On the other hand, Rubaschkin (’07, 712) and Swift (14)
have strongly supported the theory, advanced by Nussbaum
(80, ’01), that the primordial germ-cells do not originate in the
germinal epithelium, but come from cells which have not given

eh

CELLS LINING THE PERITONEAL CAVITY 421

up their embryonic character and have not been differentiated
into any especial somatic structures. Eigenmann (’91), Beard
(04), and Hoffmann (’92) have also supported this view. Swift
(15) derives the primordial germ-cells from a particular part of
the germ-wall entoderm at the edge of the area pellucida during
the primitive-streak stage. When this area becomes vascularized
by the extension of the mesoderm, these cells gain entrance to
the circulation and settle out in the region of the developing
gonad. He agrees that the medullary cords are formed from
downgrowths of the germinal epithelium, but considers the cords
of the second proliferation as formed by rapidly proliferating,
primordial germ-cells or oogonia. Finally, the oogonia form
the definitive ova, while the cells derived from the germinal
epithelium present in the cortical cords become follicular
epithelium.

A third group of workers have assumed that there are two
sources for the definitive ova, one the primordial germ-cells
which furnish a few ova, while a second generation of germ-cells
develop from the germinal epithelium. This view has been
supported by Felix (06), Allen (’04), Dustin (’07), and particu-
larly by Firket. Firket (14, ’20 b) has described the early
stages in the chick and rat, and in both species he finds the total
number of the primordial germ-cells far too few to permit of
their being considered the sole source of definitive ova, so that
he is forced to assume an additional development of ova from
the germinal epithelium. Using his two species as types of the
bird and mammal, he suggests that this process may be a phy-
logenetic recession.

The discussion of the origin of the definitive ova from the
germinal epithelium deals almost entirely with changes which
take place during embryonic life, and hence, in mammals at
least, would be most difficult to examine by the vital staining
technique. However, it is well known that in some species there
is formation of ova after birth, and in these it seems that the
vital staining of the germinal epithelium might prove a useful
adjunct to the study of the question regarding the origin of the
definitive ova. My experiments, being confined so far to the

422 R. S. CUNNINGHAM

rabbit, do not offer any assistance in settling this point. On
the other hand, the similarity between the reactions displayed by
the cells of the germinal epithelium and the general serosal lining
cells towards vital dyes suggest a closer relationship between
these two groups of cells than has usually been assumed.

SUMMARY

1. The serosal lining cells have been found to store vital dyes
in a very characteristic manner. The two most striking mani-
festations of this reaction consisted in the localization of a con-
centration of dye-granules in a circumscribed area of the cyto-
plasm of each cell and in the formation of a perinuclear rosette.

2. Mesothelial cells from different areas of the peritoneal sur-
face presented certain peculiarities in their reactions to vital
dyes which sufficed to classify them into groups, while they
still conformed to the general characteristic type.

3. The variations noted in the cells from different areas of the
peritoneal surface consisted in differences in the amount of
dye stored, the characteristics of the perinuclear rosette, and
the orientation within the cell of the localized collection of dye-
particles. The cells covering the intestine usually contained the
least amount of dye, while those of the splenic mesothelium and
the germinal epithelium of the ovary contained the largest
amount.

4. The germinal epithelium was found to store vital dyes in an

especially characteristic manner. Each cell contained a round,
oval, or cup-shaped mass of granules in the infranuclear zone
of the cell; this mass, in well-stained animals, filled the entire
portion of the cell between the nucleus and the basement mem-
brane. On the other hand, the perinuclear rosette was found
only rarely in the cells of the germinal epithelium.

a,

CELLS LINING THE PERITONEAL CAVITY 423

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424 R. S. CUNNINGHAM

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CELLS LINING THE PERITONEAL CAVITY 425

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PLATE 1

EXPLANATION OF FIGURES

1 Section of the germinal epithelium of a rabbit which had received eight
intravenous injections of trypan blue.

2 A binucleate mesothelial cell from the diaphragm of a rabbit which had
received twelve intravenous injections of trypan blue.

3 and 4 Mesothelial cells from the spleen of a rabbit which had received
ten intravenous injections of trypan blue.

5 <A mesothelial cell from the omentum of a rabbit which had received six
intravenous injections of trypan blue.

6 A mesothelial cell from the spleen of a rabbit which had received twelve
intravenous injections of carmine.

7 A mesothelial cell from the spleen of a rabbit which had received eighteen
intravenous injections of carmine.

8 Mesothelial cells from the diaphragm of a rabbit which had received twenty-
five intravenous injections of carmine.

426

CELLS LINING THE PERITONEAL CAVITY

R. 8. CUNNINGHAM

Fig 3 (x 925)

Fig.4 (x 925)

Fig. 7 (x1300)
427

PLATE 1

Fig. 8 (x1300)

Resumen por el autor, Raymond M. Selle.

Cambios en el epitelio vaginal del conejillo de Indias durante el
ciclo éstrico.

El ciclo éstrico del conejillo de Indias presenta cuatro periodos
o estados bien definidos, ademas del intervalo, que corresponde
a estados semejantes de la rata. En el primer estado, que en
apariencia no ha sido notado por Stockard y Papanicolaou, el
frote vaginal contiene solamente grandes células epiteliales granu-
losas y vacuolares. Estas células epiteliales derivan de las capas
superficiales de la mucosa, debajo de las cuales ha tenido lugar
la cornificacién. La mucosa aleanza su mayor altura en este
periodo. En el estado 2, las células epiteliales superficiales han
desaparecido, dejando como capa mas externa el estrato cérneo.
Este ultimo mediante descamacién susministra las células
tipicas, anucleadas, en forma de copos, del frote. Hasta este
momento la semejanza con la rata es muy estrecha. Sin em-
bargo, en el conejillo de Indias toda la capa cornea, no todo el
epitelio, con algunas de las células no cornificadas pueden ser
expulsadas en masa. Mientras que en el estado 3 de la rata el
material granular y caseoso consta de elementos del estrato
cérneo, descamado rapidamente, aisladamente o en pequenos
grupos, hasta desaparecer por completo, en el conejillo de Indias
la masa caseosa descrita por Stockard y Papanicolaou esta
constituida por las células epiteliales no cornificadas situadas
mas profundamente, las cuales se desprenden en grandes ntimeros.
En el ultimo estado (4) los leucocitos desecritos por Stockard y
Papanicolaou en su estado 3 aparecen. El autor no ha hallado
estado alguno que corresponda a su estado 4. No existe prueba
alguna sobre la importante contribucién del Utero a los ele-
mentos celulares de la vagina, pues la ultima es practicamente
idéntica en los conejillos normales y en los histerectomizados.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MAY 22

CHANGES IN THE VAGINAL EPITHELIUM OF THE
GUINEA-PIG DURING THE OESTROUS CYCLE

RAYMOND M. SELLE

THREE PLATES (ELEVEN FIGURES)

INTRODUCTION AND LITERATURE

The last few years have witnessed a considerable advance in
our knowledge of the cyclical changes in the reproductive organs
of the mammalian female, chiefly as the result of the introduction
of a new method by Stockard and Papanicolaou; a method by
which the course of the cycle may be followed in the living animal.
The results obtained during the preceding years involved the
sacrifice of animals at various times with relation to some definite
event such as parturition or heat (when the latter is easily de-
termined) and the microscopic study of sections. Obviously, it
was not possible to study successive cycles in the same animal.

Nevertheless, data of some importance have been accumulated
from the time of Lataste, Morau, and Retterer to the present.
Since the literature of the period is dealt with more completely
by Long and Evans (’22), it is unnecessary here to do more than
eall attention to those papers dealing more directly with the
guinea-pig and the development of the method.

As early as 1892, Retterer observed that regular changes
occurred in the vaginal mucosa of non-pregnant guinea-pigs.
Following ‘parturition he killed animals at regular intervals and
was the first to describe a stratum cornium in the guinea-pig,
although such a layer had been described in mice by Morau in
1889. He found that on the fifteenth day postpartum a layer of
cornified cells was beginning to form under the superficial layers
of the stratified vaginal mucosa.

In a number of papers Leo Loeb reported his results on the
study of ovulation and the formation of corpora lutea in guinea-
pigs. He came to the conclusion that it recurred at intervals of

429

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, No. 4

430 RAYMOND M. SELLE

about twenty-one days. Similar investigations on the incidence
of ovulation, ete., in rats and mice were being carried on in this
laboratory by Doctor Long and several students.

No further work of importance on the vaginal cycle of the
guinea-pig was recorded until 1917, when Stockard and Papani-
colaou published their paper on “The existence of a typical oes-
trous cycle in the guinea-pig with a study of its histological and
physiological changes.”’ They studied the vaginal cycle by tak-
ing samples of the contents of the vagina at regular intervals.
They obtained these samples by introducing a small nasal specu-
lum into the vagina, the arms of which were held apart by means
of a thumb-screw, allowing them to observe the entire lumen of
the vagina. They were thus able to recognize an oestrous rhy-
thm consisting of four stages, each having a characteristic vaginal
fluid which contained cells from the walls of the vagina.

The vaginal fluid during the first period contained a great
amount of mucous secretion, many desquamated epithelial cells,
and non-nucleated, or cornified cells, which appeared toward the
end of the stage. The second stage could be recognized by the
thick, cheese-like contents of the vagina due to the accumulation
of the desquamated epithelial cells. During the third stage the
vaginal fluid became thinner because of the solvent action of
leucocytes which invaded the epithelium and entered the lumen.
The fourth stage, during which a small amount of blood was found
in the vagina, was of short duration and did not always occur.
These stages were followed by the dioestrum, or intermenstrual
period, during which the vaginal fluid contained mucus, atypical
squamous cells, and many leucocytes. They then correlated
these stages with the conditions of the uterus and ovary at cor-
responding periods.

A second paper by Stockard and Papanicolaou appeared in
1919 in which they described the vaginal closure membrane and
the time of copulation in relation to the oestrous cycle. They
found that there was formed regularly a very delicate epithelial
membrane which closed the orifice of the vagina soon after the
‘heat period’ and remained closed until the next cycle or until
parturition in case of pregnancy.

VAGINAL EPITHELIUM OF GUINEA-PIG 431

As the result of following the suggestions contained in Stockard
and Papanicolaou’s earlier paper, the cycle in the rat was worked
out (Long, 719) and later elaborated and verified (Long and
Evans, ’22). Smears taken from the vagina of the rat by means
of a small spatula were studied and correlated with sections of all
parts of the reproductive system. By means of the smears, it
was possible to recognize four definite stages besides the interval.
Smears taken during the first stage contained nucleated epithelial
cells of uniform size; during the second stage, few, large, cornified
cells; in the third stage they were made up of a great abundance
of cornified cells; in the fourth stage, by many leucocytes admixed
with cornified cells. Throughout the interval between stage 4
and stage 1, or the dioestrous pause, the vaginal fluid contained
leucocytes with only a few epithelial cells.

The sections revealed a cyclical growth and desquamation of
the vaginal epithelium. The epithelium is lowest during the
interval, increasing in thickness just before the advent of stage 1.
One of the striking features is the occurrence, during this stage,
of the processes of cornification not in the superficial layers but
at a depth of almost two cells, the upper two layers of cells retain-
ing their epithelial character, later to be shed as the cells of the
smear of stage 1. The cornified stratum so denuded becomes
superficial, increases in thickness, and is later detached, thus
furnishing the cells of stages 2 and 3. Leucocytes, of which the
epithelium is devoid during stages 1, 2, and 3, now invade the
mucosa and escape into the lumen in stage 4.

Long and Evans attempted to correlate the cycles in the rat
with the cycles in the guinea-pig as found by Stockard and
Papanicolaou and as observed by themselves in twenty-two
guinea-pigs for a couple of months. In a section of the vagina
of the one guinea-pig which they killed, Long and Evans ob-
served what they believed to be the cornified layer under a layer of
epithelial cells. Since they found that such a condition occurred
regularly in the rat, they conjectured that an epithelial layer
superficial to the cornifying layer must be normal in the guinea-
pig, and if such a condition were true, then it was evident that
Stockard and Papanicolaou had overlooked it.

432 RAYMOND M. SELLE

With these results in mind, it seemed worth while to study the
changes in the vaginal epithelium of the guinea-pig in order to
ascertain whether or not a layer of epithelial cells occurred super-
ficial to the stratum cornium as observed by Retterer; to supple-
ment the work of Stockard and Papanicolaou, and to correlate
the rat and guinea-pig more exactly. The problem was under-
taken at the suggestion of Prof. J. A. Long and was carried out
under his general direction in the Zoélogical Laboratories.

METHODS

The guinea-pigs chosen for these experiments were common
white, brown, and mixed colored virgin females about a year old.
They were kept at a relatively constant temperature averaging
68° to 72°F. and were fed a constant, daily ration of green grass or
alfalfa hay, carrots, and rolled barley, because there is reason to
believe that a lower or a higher temperature as well as variation
in the ration tends to affect the span of the oestrous cycle.

Since the determination of the state of progress of the cycle,
as shown by the previous work cited on the guinea-pig and rat,
is dependent on our knowledge of the character of the contents of
the vagina, the method of sampling the contents becomes a mat-
ter of some importance. Several methods of taking samples were
employed. For reasons explained later, the method employed
by Stockard and Papanicolaou was discarded, and because of
the extreme length of the vagina in the guinea-pig the spatula
method as used by Long and Evans on the rat was not of much
value.

The pipette method, suggested by Doctor Long and developed
by Miss E. Fisher in this laboratory for making vaginal smears
from mice, was tried, and after several improvements a satisfac-
tory method for taking samples was perfected. The instrument
used was made by inserting into a 25-ec. oval, rubber bulb a piece
of thin-walled, glass tubing 14 cm. long and 1 cm. in diameter,
about 5 em. of which was drawn down to a diameter of 5mm.

When a sample was to be taken, the animal was held ventral
side up in the left hand allowing it to lie on its back along the
left forearm with its head toward the elbow. The left thumb

ii i

VAGINAL EPITHELIUM OF GUINEA-PIG 433

was placed in front of the animal’s left hind leg and exerted
pressure just in front of the vulva; at the same time with the left
forefinger between the vulva and the right leg, it was easy slightly
to open the orifice of the vagina. The syringe, containing about
1 ec. of warm, normal, salt solution, was then introduced into the
vagina to a depth of 25 to 40 mm. By squeezing the bulb and
forcing the contents of the syringe into the vagina once or twice,
it was possible to get a characteristic sample of the cells in the
lumen. If, while withdrawing the syringe, pressure was applied
anterior to the vulva with the thumb of the left hand, the sample
could be drawn up readily into the syringe. Enough of a sample
was obtained at one operation by this method to make several
smears. This proved a most satisfactory method, because each
time a sample was taken the vagina was washed out and the cells
which were free in the lumen were removed; consequently, it was
reasonable to suppose that all of the cells which were found in any
one sample had been shed during the interval immediately follow-
ing the preceding sample.

In order to study the cycle, samples were taken at 8 A.M., 12 M.,
4 p.m., and 8 p.m., and the results recorded. Animals were then
killed during each stage as revealed by the character of the smears.
A small amount of 0.75 per cent solution of table salt immediately
followed by Bouin’s fixing fluid was injected under pressure pos-
teriorly into the aorta just anterior to the diaphragm. By this
method the vagina in situ was fixed in its normal position at the
earliest possible moment and its excision greatly facilitated.
After removal from the body the vagina was immersed twenty-
four hours longer in the fixing fluid. Most of the picric acid was
extracted in 50 per cent alcohol containing lithium carbonate.
Portions of the vagina including the cervix were then dehydrated,
cleared, and imbedded in paraffin and sectioned at 7jy. The
condition in the vaginae as revealed by these sections was then
correlated with the smears taken just before killing the animal.

The uterine glands in the guinea-pig secrete an abundance of
mucus which flows down into the vagina (Stockard and Papani-
colaou, 717), often obscuring the nature of the smear. Indeed,
Stockard and Papanicolaou found uterine epithelial cells thus

434 RAYMOND M. SELLE

carried into the vagina. In order to avoid any confusion that
might be caused by the presence of uterine mucus and cells,
in seven of the twenty females the uterine horns were tied off
posterior to the oviducts and anterior to the cervix and the inter-
mediate pieces excised. ‘The value of this operation was not
sufficient to warrant its recommendation. Although the con-
tents of the vaginae in the animals which had been hysterecto-
mized were freer from mucus and consequently more easily diag-
nosed, there was not a great difference between smears made
from these animals and smears made from normal guinea-pigs.
A careful scrutiny of successive vaginal smears of twenty
guinea-pigs over a period of seven months disclosed a succession
of cell changes in the contents of the vagina substantially similar
to that described by Stockard and Papanicolaou for the guinea-
pig and by Long and Evans for the rat. The enumeration of the
stages in this paper follows that employed by Long and Evans.

CHANGES IN VAGINAL SMEARS AND HISTOLOGY OF VAGINAL
EPITHELIUM

Interval of dioestrum

If the vaginal smears of a number of guinea-pigs were to be
examined, it would be found that most of them would contain
many leucocytes and few, small, round epithelial cells; in fact,
this condition would be found to be characteristic during three-
fourths of the time in any one pig. While the nature of the smear
does not seem to vary during this period there are, however, his-
tological changes in the mucosa.

The lining of the vagina of a guinea-pig consists of a stratified
epithelium, the upper surface of which is nearly even, while the
side in contact with the submucosa, from which it is clearly de-
marked, appears insection to be deeply lobed as though furrowed,
the effect being produced by tongue-like projections of the sub-
mucosa extending up into the epithelium. The epithelium thus
seems to vary in thickness (figs. 1 and 8). The whole mucosa is
further thrown into larger longitudinal folds.

ove = 4 a

age ee

VAGINAL EPITHELIUM OF GUINEA-PIG 435

At about the middle of the interval the epithelium was found to
be thinner than at any other time, being only one or two layers
of cells high at the thinnest places. At the deep ends of the down-
ward projections of the epithelium were numerous mitoses—a
condition which apparently substantiated Stockard’s and Papani-
colaou’s statement that the vaginal epithelium was regenerated
from the base of the infoldings.

Towards the end of the interval the epithelium rapidly in-
creased in height until it reached a maximum of ten to twelve
cells. The uppermost of these cells which bordered the lumen
had become enlarged and vacuolated, with their nuclei usually
at their bases. The cells lining the inner surface of the vagina
were the most vacuolated, while the layers beneath were smaller
and gradually approached the normal size of the epithelial cells
at about three or four layers below the surface (figs. 2 and 9).

Stage 1. Large irregular epithelial cells only
(No corresponding stage described by Stockard and Papanicolaou)

Stage 1 marks the beginning of the oestrous cycle. A smear
made early in this stage exhibited, in marked contrast to that
of the interval, first, numerous, large, round or odd-shaped, epi-
thelial cells containing many vacuoles, and, secondly, very few
leucocytes. The latter undoubtedly remained over from the
interval which, as just described, was characterized by the pres-
ence of many leucocytes and a very few epithelial cells. These
epithelial cells appeared in the smear singly or in groups of three
or more.

A section of the mucosa early in stage 1 showed the epithelium
to be practically as high as at the end of the interval, i.e., about
ten to twelve cells; also that a characteristic transformation had
already made its appearance. ‘This transformation was exhibited
in cornification of certain of the epithelial cells as shown in
figures 3 and 10. It will be observed that the most superficial
cells were enlarged, were odd-shaped, and contained vacuoles;
that the next deeper cells were somewhat smaller and more
spherical, and that the next deeper levels showed the beginning

436 RAYMOND M. SELLE

of the process of cornification. The former of these were the cells
which were cast off and furnished the cells found in a sample
taken during this stage. They are called superficial epithelial
cells for the reason that they were above a region in which corni-
fication later took place. The cornifying process began rather
suddenly and continued at a rapid rate simultaneously through-
out the entire vagina. It began two to four cells below the sur-
face of the superficial epithelium, and involved four to six cell
layers. In some eases it extended up into the superficial epithe-
lial cells before the latter had been entirely cast off. This is
shown in figure 4. Beneath the stratum cornium may be dis-
tinguished the typical layers of stratified epithelium.

A little later in this stage the leucocytes had entirely disap-
peared from the vaginal fluid and the only cells found in the
lumen were the superficial epithelial cells. As a result of the
sloughing off of these cells, the vaginal mucosa became reduced
in height and the cornified layer exposed.

It usually happened that the shedding of these superficial
epithelial cells occurred while the entrance to the vagina was
closed by the vaginal closure membrane described by Stockard
and Papanicolaou, often making it necessary to break the mem-
brane in order to get a sample.

Stage 2. Cornified cells only
(Stockard and Papanicolaou’s stage 1)

A vaginal sample taken during this stage contained many
flattened, horny, non-nucleated or cornified cells. No leucocytes
were found in the lumen. Stockard and Papanicolaou in their
first paper mentioned two kinds of cornified cells. These are
apparently two different stages of the same process of cornifica-
tion. The various intermediate degrees of cornification can be
demonstrated both in smears and sections counterstained with
eosin, the amount of stain taken by the cells varying with the
extent of cornification.

Because of the fact that there were almost always a number of
cornified cells at the external orifice of the vagina, smears of the

VAGINAL EPITHELIUM OF GUINEA-PIG 437

succeeding stages were constantly contaminated by them, es-
pecially in those obtained by means of a nasal speculum or a
spatula; hence the value of the syringe method.

As the process of cornifiecation continued, the cornified cells
became loosened from the underlying layers and were cast off
into lumen singly or in flakes of many cells.

An interesting occurrence not described by any writer was the
shedding of the entire cornified layer as a cast which showed the
natural shape of the lumen of the vagina. It was possible in
several animals by the use of the syringe to get several succeeding
cornified casts at regular intervals of sixteen days. When pieces
of the casts were examined under a microscope, the structure was
readily recognized as continuous layers of superimposed cornified
cells. Some very perfect casts were obtained, even showing the
folds about the cervix. Fourteen complete cornified linings or
casts were obtained and preserved, besides a great many more
casts broken in the process of taking the sample. This shedding
of the entire lining of the vagina occurred at the end of stage 2.

The shedding of the entire cornified layer would seem to have
been mistaken by Stockard and Papanicolaou to be the whole
vaginal epithelium separated from the underlying connective tis-
sue, for, according to them (719, p. 234), “the epithelium is now
expelled as one continuous tube forming the cover around the
vaginal plug instead of sluffing off in smaller pieces as occurs dur-
ing the fourth stage when copulation has not occurred. However,
the vaginal epithelium may occasionally be shed en masse without
copulation. . . . . Itisclear, therefore, that what was termed
by Lataste the ‘enveloppe vaginale’ is the layer of epithelium
separated from the underlying connective tissue. ae

That the entire vaginal epithelium did not separate froin: the
connective tissue, but that only the whole cornified layer became
loosened from the underlying epithelium and lay free in the
lumen, is shown in figure 5. The condition of the vaginal epi-
thelium immediately after the shedding and expulsion of the
cornified cast is shown in figures 6 and 11.

438 RAYMOND M. SELLE

Stage 3. Cornified cells and small epithelial cells

The characteristic smear for this stage consisted of cornified
cells, and of nucleated, small, epithelial cells. There are as yet
no leucocytes found, but as the stage progressed fewer cornified
cells and increasing numbers of epithelial cells appeared. Some
of the latter contained granules in the cytoplasm. In case the
cornified layer had been shed in a cast, this stage lasted only a
short time during which the epithelial cells were cast off.

The active shedding of these two kinds of cells reduced the
height of the stratified epithelium to from four to seven cells.

This stage was called the cheesy stage by Stockard and Papani-
colaou, because of the appearance of the mass of epithelial cells.
Since in this investigation the vagina was regularly washed out
with warm saline solution in the process of taking samples and
was thereby kept clean, the cells could not accumulate to form
such cheesy masses.

During this stage leucocytosis began in earnest. Leucocytes
invaded the submucosa and the epithelium, in the latter of which
it was common to see aggregates of three to ten leucocytes. In
many cases leucocytes had become imbedded in the epithelial
cells, as many as five having been counted in a single cell. Corni-
fied cells had completely disappeared from the smears and did not
appear again until the next cycle.

Stage 4. Small epithelial cells and leucocytes

(Stockard and Papanicolaou’s stage 3)

This marks the advent of leucocytes in the lumen of the vagina.
In the previous stage the leucocytes were migrating into the
vaginal epithelium, but had not yet reached the lumen. Smears
made during stage 4 contained the nucleated epithelial cells of
the preceding stage and also increasing numbers of polymor-
phonuclear leucocytes. At first the epithelial cells predominated,
but gradually ceased to become detached; while at the same time
the number of leucocytes rapidly increased until they were in a
majority.

At the end of this stage the epithelium was only two to four
cells high over the finger-like processes of the submucosa (fig. 7).

— —

VAGINAL EPITHELIUM OF GUINEA-PIG 439

Following this stage, Stockard and Papanicolaou described
another, which they called the fourth, during which blood was
found in vaginal samples. In these investigations only two cases
were observed in which blood was found in the smears, and in
both cases it occurred at the end of stage 4. Itis hardly probable
that this is a common occurrence. Because of the thin epithelial
covering over the submucosa at this stage, it seems highly prob-
able that an instrument inserted into the vagina might injure
some of the many blood vessels in the submucosa and thus
permit blood to escape into the vagina.

Because of the differences in the stages of the cycles of the
guinea-pig as found by Stockard and Papanicolau and in these
investigations, the following table (1) has been constructed in

TABLE 1

A comparison of the stages in the oestrous cycles in the guinea-pig and rat

GUINEA-PIG
(STOCKARD AND PAPANICOLAOU)

GUINEA-PIG (NEW)

stage
Ae
First period:
Squamous epithelial cells

Second period:
Cornified cells

2.
Cheesy mass, epithelial
cells

3.
Leucocytes and epithelial
cells

4.
Appearance of blood in
lumen

5.
Interval:
Leucocytes and epithelial
cells

RAT (LONG AND EVANS)

stage
ils
Large irregular epithelial
cellsonly. (Superficial
epithelial cells)

2.
Cornified cells only

3.

Cornified cells and small
epithelial cells, or small
epithelial cells only

4,
Leucocytes and epithe-
lial cells

5.
Interval:
Leucocytes (few epithe-
lial cells)

stage
ibs
Superficial epithelial
cells only

9

a“

Cornified cells only

3:
Many cornified cells
(cheesy)

4,

Leucocytes and cornified
cells (sometimes epi-
thelial cells)

a:
Interval:
Leucocytes and epithe-
lial cells

440 RAYMOND M. SELLE

order to point out the similarities as well as the differences. It
will be seen that these newly determined stages in the guinea-pig
correspond more closely to stages in the rat as described by Long
and Evans. ©

Interval

At the end of stage 4 or at the beginning of the interval the
vaginal closure membrane began to form as described by Stockard
and Papanicolaou, growing very rapidly and, if not disturbed,
closing the vagina in one to three days. This membrane remained
intact until mechanically destroyed or until the next cycle,
when it was normally broken during the cornified stage by the
tension produced by the swollen vulva and accumulated fluid in
the lumen. When samples were taken daily, the membrane was
prevented from forming for an indefinite period.

It will be recalled that toward the end of stage 4 and the be-
ginning of the interval the rapid rate of desquamation of the
epithelial cells gradually diminished, while the leucocytes in-
creased until they were in the majority. The transitional period
between stage 4 and the interval is indicated by the decreasing
numbers of epithelial cells and increasing numbers of leucocytes
in the smears. Samples taken up to the middle of the interval
contained a few epithelial cells, but for the next week or more only
leucocytes. From this time on until the beginning of stage 1
vaginal smears were found to exhibit leucocytes and some atypical
cells. ;

A section through the vagina during the middle of the interval
showed the epithelium to be only one or two cells high. This
reduction in height was no doubt caused by the continued drop-
ping off of the epithelial cells which were found in the vaginal
fluid following stage 4.

Toward the end of the interval and immediately before the
beginning of stage 1 the epithelium was rebuilt to a height of ten
to twelve cells (fig. 2). The uppermost of these cells became
vacuolated as described under the interval at the beginning of the
discussion on stages.

It is interesting to note that, although there was the above
rapid transformation in the epithelium, such a condition could

lati gl pe ———

VAGINAL EPITHELIUM OF GUINEA-PIG 441

not be suspected by a mere study of the smears. . Consequently,
the method of obtaining material for histological study at this
critical time involved a departure from the ordinary procedure.
In the ordinary procedure it was only necessary to keep records
of smears made from the animals under operation and to kill the
pig at the desired period in order to correlate the smear with the
condition in the vagina as shown in sections. In the above ex-
ception, the one killed for the high epithelium shown in figures 2
and 9, the method used was to study the length of consecutive
cycles in all the animals and then to select the animal with the
most regular recurring cycles and to kill it twenty-four hours be-
fore the calculated cornified stage.

In this connection the question arises, may not frequent
douching of the vagina with warm saline solution affect the length
of the cycle, and also may not the operation for hysterectomy
affect the following cycles in the animal. By referring to table
2, the following facts will be made clear. The average length
of the one hundred eleven cycles observed in twenty-four guinea-
pigs was found to be 15.87 days. This corresponds to the length
of the cycle as found by Stockard and Papanicolaou who found
the average length of the cycle to be 15.73 days for sixty-seven
cycles. There was no perceptible difference in the length of
eycle in the normal and hysterectomized animals (table 2).

SUMMARY

1. The syringe method for taking vaginal samples is more
satisfactory than any other method yet described for the guinea-.
pig.

2. The length of the oestrous cycle of the guinea-pig was found
to be 15.87 days, the same as determined by Stockard and
Papanicolaou (15.73). ;

3. The oestrous cycle has four well-defined periods or stages
besides the interval.

4, Stage 1. The epithelium is at its greatest height at the be-
ginning of this stage, ten to twelve cells. Cornification has been
going on beneath the superficial layers. Vaginal smears contain
large, vaculated, granular, odd-shaped, epithelial cells.

442

RAYMOND

M. SELLE

5. Stage 2. This is the stage of desquamation of the flattened,
scale-like, non-nucleated cells—cornified cells. The inner corni-
fied portion of the vaginal mucosa loosens and may be shed as a

cast.

TABLE 2

Showing the length of consecutive cycles in twenty guinea-pigs from August, 1920, to
January, 1921

AVERAGE
ANIMAL LENGTH OF CONSECUTIVE CYCLES IN DAYS LENGTH OF
CYCLE
199 16; TGS Wig LG May 16.50
200! 15, 14, 15 14.66
2061 1516; 16216; 1G 7s lop6 15.75
208 (LO 1 5seks, LO okie veal Ornlo 16.22
211 162.16" 16; 16) 17, S16 16.00
213! V7ianG 16.50
220 15, 16, 16 15.66
232 15, 15 155; lo; Lo, ald 15.42
233 16; 17; 16 16.33
246 14, 14, 16, 16, 16, 16, 16, 15 15.37
262! 15,16, 15,16, 16; 15; lo Ose (ers 15.66
Tipe 17, 16 16.50
277 ANG leg 16.50
279 16, 16 16.00
280 15, 16; 15, 16; 167 16, 15, 15, 15; 15 15.40
281! 16 16.00
282 17,06, 11% 16.66
285 1G GS he TO Vie eie aon 16.44
2961 17. Toy 1G. AG. 6. 6) 16 alos to, 16 16.00
298 16) 15; 15,9 145515; ib; 6 15.28
INfumbenxotal4-cayvaevcles san rie aliens 1: venkaeeRae etchant rar 4
Number orn 5-dayeGycless 5 . kat yaad itis tect. 9c chee ste tae oy -Peraetan ota toha tet 28
Nim bex tate 1 Gay" Cy Chess. ers cto tact et aetotan ns © hs evsuel ithe hers nets mers ae 57
Numibertor- 17-day cy clesel hain: tet er teste <tocieters ss ude ia e hres taanlnneat 22
Totalkmumber Ol Cy Clestit antes nck ea at oe trap ate eials whee. whi mtayorelp late eae 111
Wiodetss tae: Ot wile Sa ee ge etn: Meee Wh Lace amend Ry iter 16.0 days
Goeneraliawerager.(. Aare eee ccs mane. gaat ian ae oie as 15.87 days

1 Animals having both horns of the uterus excised.

6. Stage 3.

Vaginal smears made during this stage contain
round, nucleated, epithelial cells and some cornified cells which
have remained from the preceding stage. Leucocytosis begins
during this stage but leucocytes do not enter the lumen.

VAGINAL EPITHELIUM OF GUINEA-PIG 443

7. Stage 4. During this stage leucocytes appear in the lumen
of the vagina for the first time since the beginning of the cycle.
Smears contain epithelial cells and leucocytes.

8. Stage 5 or interval. Vaginal smears contain leucocytes and
mucus, but very few or no epithelial cells. The epithelium has
become reduced to its lowest condition, one to two cells. The
epithelium is rapidly regenerated at the end of the interval
immediately preceding the next cycle.

9. Stage 4 of Stockard and Papanicolaou, marked by the ap-
pearance of blood, is very doubtful.

BIBLIOGRAPHY

Iso, O. 1920 Observations on the sexual! cycle of the guinea-pig. Biol. Bull.,
vol. 38, no. 4.

Larasts, F. 1892 Transformation périodique de l’epithelium du vagin des
rongeurs (rhythme vaginal). C.R. Soc. Biol., T. 44, pp. 765-769.
1893 Rhythme vaginal des mammiféres. C. R. Soc. Biol., T. 45,
Memoirs, pp. 135-146.

Lors, Leo 1909 Uber die Bedeutung des Corpus luteum. Zentralbl. f. Phy-
gO, I8Cl, SB, Se (eae
1910 Weiterer untersuchungen iiber die kiinstliche Erzeugung der
miitterichen Placenta und iiber diemechanik des sexuellen Zyclus des
weibichen Langethierorganismus. Ibid., Bd. 24, S. 203-208.

Lone, J. A. 1919 The oestrous cycle in rats. Anat. Rec., vol., 15.

Lona, J. A., anp Evans, H. M. 1920 The oestrous cycle in the rat and other
studies in the physiology of reproduction. Anat. Rec., vol.18. 1921.
Further studies on the physiology of reproduction. Anat. Rec.,
Violen le
1922 The oestrous cycle in the rat and its associated phenomena.
Univ. of California Memoirs.

Marsan, F. H. A. 1910 The physiology of reproduction.

Morav, H. 1889 Des transformations epithéliales de la muqueuse du vagin
de quelques ronguers. Jour. del’Anat. et dela Phys., 1889, pp. 275-297.

ReEtTTeERER, E. 1892 Evolution de l’épithélium du vagin. (Deuxiéme
note.) Memoires Comptes Rendus Société de Biologie, Paris, T. 44, pp.
566-568.

1892 Sur les modifications de la muquése utérine a l’époque du rut.
Com. Ren. Soe. Biol., T. 44, pp. 637-642.

StTocKArD, C. R., anpD PapanicoLtaou, G. N. 1917 The existence of a typical
oestrous cycle in the guinea-pig with a study of its histological and
physiological changes. Am. Jour. Anat., vol. 22.

1919 The vaginal closure membrane, copulation, and the vaginal
plug in the guinea-pig, with further considerations of the oestrous
rhythm. Biol. Bull., vol. 37.

PLATE 1

EXPLANATION OF FIGURES

1 (Microphotograph.) Longitudinal section of the vagina at the middle
of the interval, showing the finger-like processes of the submucosa projecting up
into the epithelium. The submucosa contains many blood vessels. The epithe-
lium is thinnest at this time, being only one or two cell layers. %X 115.

2 (Microphotograph.) Longitudinal section of the vagina near the cervix
at the end of the interval, about twenty-four hours before stage 1, showing the
epithelium at its greatest height, about ten cells. The uppermost cells are
greatly enlarged and vacuolated and are the superficial epithelial cells found in
vaginal smears during stage 1. Leucocytes are seen in the lumen. X 115.

3 (Microphotograph.) Longitudinal section of the vagina during stage 1.
Some. of the large irregular epithelial cells characteristic of stage 1 are seen in
the lumen along with a few leucocytes. The uppermost layers of cells have begun
to slough off. About three or four cells below the surface of these superficial
epithelial cells the cornifying layers are seen as flattened and more darkly stained
cells. XX 115.

4 (Microphotograph.) Longitudinal section of the vagina during stage 1,
showing the well-formed cornified layers splitting from the stratum granu-
losum below and the process of cornification extending up into the superficial
epithelial layers above. X 115.

444

OE

PLATE t

‘
x

LIUM OF GUINEA-PIG

VAGINAL EPITHE

SELLE

RAYMOND M.

415

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, No. 4

PLATE 2

EXPLANATION OF FIGURES

5 (Microphotograph.) Transverse section of the vagina near. the cervi x
showing the cornified layers free in the lumen. The animal was killed at
beginning of stage 2. Notice the few superficial epithelial cells between
two cornified layers. X 115. -

6 (Microphotograph.) one tt ieee section of the vagina near the ¢
showing the condition of the epithelium immediately after the entire cor
layer was shed asacast. The epithelium is only four to seven cells high. §
Saas. Ea

7 (Microphotograph.) Longitudinal section of the vagina near the
showing the condition of the epithelium during stage 4 when epithelial
leucocytes are found in the lumen. The epithelium is about three to:
high. X 115. -

2

7
uu

PLATE

SA-PIG

UINE

‘
x

PITHELIUM OF G

RAYMOND M.

VAGINAL E

SELLE

=; .
Og

*
-

“2 @

aa

eee

447

PLATE 3
EXPLANATION OF FIGURES

8 Part of mucosa in figure 1 enlarged. Middle of interval. > 624.

9 An enlarged portion of the epithelium in figure 2. End of interval. Note
the enlarged superficial epithelial cells. X 572.

10 The upper part of the mucosa in figure 3 more highly magnified. Stage 1.
The superficial epithelial cells are vacuolated. Under them is the beginning
of the cornified layer. > 624.

11 More highly magnified portion of epithelium in figure 6. After the shed-
ding of the cornified layer. Stage 3. X 624.

(Drawn by Miss Edna Fisher)

448

VAGINAL EPITHELIUM OF GUINEA-PIG PLATE 3
RAYMOND M. SELLE

Resumen por el autor, Ivan E. Wallin.
Sobre la naturaleza de las mitocondrias.

Ill. La demonstracién de las mitceondrias mediante los métodos
bacteriol6gicos.

El] autor ha preparado frotes de tejidos animales en porta-
objetos. Las mitocondrias se tinen con los colorantes bacterio-
logicos en estas preparaciones.

IV. Un estudio comparativo de la morfogénesis de las bacterias
de los nédulos de las rafces y de los cloroplastos.

El Bacillus radicicola experimenta un desarrollo morfol6gico
en los nédulos de las raices, semejante a la morfogénesis de los
cloroplastos. Una nueva forma de bacteria ha sido descubierta
por el autor, quien discute la simbiosis y hace notar la analogia
de la simbiosis en los liquenes con su concepto de las mitocondrias.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MAY 1

ON THE NATURE OF MITOCHONDRIA

III. THE DEMONSTRATION OF MITOCHONDRIA BY BACTERIOLOGICAL
METHODS

IV. A COMPARATIVE STUDY OF THE MORPHOGENESIS OF ROOT-
NODULE BACTERIA AND CHLOROFLASTS

IVAN E. WALLIN

Department of Anatomy and the Henry S. Denison Research Laboratories, University
of Colorado, Boulder

TWO PLATES (NINE FIGURES)

III. THE DEMONSTRATION OF MITOCHONDRIA BY BAC-
TERIOLOGICAL METHODS

In a former paper (22) the author submitted evidence to
show that the special mitochondrial technique in general use,
including the vital janus-green method, is not specific for mito-
chondria, but will also stain bacteria. While, from a purely
theoretical consideration, the properties of mitochondria are of
such a nature that one could hardly expect them to respond to
bacteriological methods, an analysis of results in this direction is
not only interesting, but also instructive.

The author has not been able to find any references in the litera-
ture to mitochondrial staining with bacteriological methods.
The results recorded below will serve to indicate not only the
reactions of mitochondria to such methods, but will also indicate
possibilities in mitochondrial manipulation.

MATERIALS AND METHODS

The tissues used in this study have consisted of various samples
from young rabbits, kittens, and mature dogs. These tissues
have included lymph nodes, liver, pancreas, kidney, salivary
glands, suparenal, thymus, and other tissues. Immediately after

451

452 IVAN E. WALLIN

removal from the previously killed animal, smears of the organs
and tissues were made on microscopical slides. The smears
were then permitted to dry in the air without any other fixation.

A large number of bacterial staining methods were later applied
to the smear preparations. Most of these staining methods have
no selective action on bacteria, and consequently the entire smear
was stained and mitochondria could not be distinguished with
any important degree of clarity. In a few instances Gram’s
stain appeared to give a little clearer differentiation. One bac-
terial staining method was found, however, that gave a sharp
differentiation—Pappenheim’s pyronin-methyl green. This stain
has had very extensive use in bacteriological technique. ‘Todd
(18) recommends it especially for the demonstration of bacteria
in cells on account of its selective action. In this study saturated
aqueous solutions of pyronin and methyl green have been used
in various proportions of mixture. In some instances it was
found that a special proportion of the two stains was necessary to
produce sharp differentiation. As Todd has recommended for
the demonstration of bacteria, it is necessary to experiment with
the proportions of the two stains to attain the best results.

RESULTS OF BACTERIOLOGICAL STAINING METHODS ON TISSUE
SMEARS

Figure 6 is a camera-lucida drawing of a part of a young
rabbit pancreas smear after pyronin-methyl-green staining.
There was only a small area in the entire smear that appeared
like the illustration. It is only in a very few cases out of a
great number of attempts that anything resembling mitochondria
were present in pancreas preparations. The bodies in the
smear, represented in figure 6, appear like mitochondria, but not
like the typical mitochondria of pancreas cells. Obviously, I
am not in a position to definitely state that these bodies are mito-
chondria. It appears probable to the author that these bodies
are the fragments of the original mitochondria of the pancreas
cells. They may be artifacts. If they are, then, what evidence
do we have of the reality of mitochondria in stained preparations?

ON THE NATURE OF MITOCHONDRIA 453

Figure 7 is a camera-lucida drawing of a part of a rabbit-liver
smear after pyronin-methyl-green staining. The small stained
bodies in this preparation appear like the typical mitochondria
of liver cells. I can find no alibi for their mitochondrial nature.

Figure 8 is a camera-lucida drawing of a portion of a rabbit-
kidney smear after pyronin-methyl-green staining. It is difficult
to find a place in the kidney smears where mitochondria-like
bodies are distinct or present. The group of kidney cells illus-
trated was more or less intact. The minute bodies in the illus-
tration were sharply differentiated and are not unlike the typical
kidney mitochondria.

Figure 9 is a camera-lucida drawing of a part of a rabbit
lymph-node smear after pyronin-methyl-green staining. The
minute bodies represented in the illustration were sharply differ-
entiated not only in the cells intact, but also in the cytoplasm of
ruptured cells. They appear like the typical lymph-node mito-
chondria. 5:

In a number of lymph-node-smear preparations it was observed
that in some cells the entire cytoplasm, which was apparently
intact, was homogenously stained with pyronin (the member of
this stain combination which stains the mitochondria). Further,
in practically all tissue-smear preparations, the ruptured cyto-
plasm is distinctly stained by the pyronin. This latter action
of the stain is interesting on account of the supposition (original
contention of Pappenheim?) that pyronin-methyl green is a
selective stain for certain lymphocytes, staining their cytoplasm
pink or red.

It appears reasonable to the author that in the cases where the
cytoplasm stains with the pyronin the mitochondrial substance
has diffused into the cytoplasm. If this interpretation is correct,
it assumes that mitochondria are composed of a substance that
is miscible with cytoplasm. It, further, assumes that under
normal conditions mitochondria have a wall, pellicle, outer
membrane, or some limiting structure. The diffuse character
of the mitochondrial substance has also been observed in
the ordinary fixed and stained mitochondrial preparations of
lymph nodes. 2

454 IVAN E. WALLIN

These illustrations, it seems to me, are sufficient evidence that
mitochondria may be demonstrated by bacteriological methods.
In this work various problems in connection with the behavior
of mitochondria have arisen. ‘These problems have no fundamen-
tal bearing on the major problem of these studies, and con-
sequently have not been pursued.

DISCUSSION

Aside from the demonstration of staining mitochondria by ’
means of bacterial technique, certain facts observed in this study
are of importance in connection with the main problem.

In a number of attempts to demonstrate the mitochondria
in adult dog tissues with the above-described bacteriological
technique, I have practically failed. Only in a very few instances
have I been able to distinguish mitochondria-like structure in
these preparations. These attempts were made with stains from
a different source than the ones originally used on kitten and
rabbit tissues. The original were Gribler’s stains. However,
with the kitten and rabbit tissues the results were not the same for
all tissues. It was exceedingly difficult to demonstrate any mito-
chondria-like bodies in the pancreas. I have not been able to
demonstrate any mitochondria in the salivary glands, thyreoid,
and suprarenal. On the other hand, it appears quite easy to
stain the mitochondria in the cells of lymph nodes. It must
be borne in mind that in all these preparations the tissues were
crushed when the smear was made.

Two prominent facts stand out in these results: first, mito-
chondria are not as delicate as it has been supposed; second,
mitochondria vary in fragility.

Regarding the delicacy of mitochondria, Cowdry (718) main-
tains that the slightest desiccation of a tissue is sufficient to
alter them. The above-recorded results with bacteriological
methods demonstrate the fact that mitochondria may retain their
form and be stained after the degree of desiccation present from
complete drying in the air.

These staining reactions, further, indicate the danger in formu-
lating hypothesis and drawing too extensive conclusions on the
basis of staining reaction alone.

ON THE NATURE OF MITOCHONDRIA 455

IV. A COMPARATIVE STUDY OF THE MORPHOGENESIS OF
ROOT-NODULE BACTERIA AND CHLOROPLASTS

In a growing conception of a bacterial nature of mitochondria,
one naturally would seek an example of an undisputed symbiotic
bacterium forstudy. The root-nodule bacteria, Bacillus radicicola
offers an example of a relationship between two organisms that is,
at least, of a partial symbiotic nature. The bacterial organism
in this case, apparently, exists as a free-living organism in the soil.
Under favorable circumstances, it enters the root hairs of Legum-
inosae and ultimately may be found in the cytoplasm of the cells
of the root-nodules. The host plant responds to the infection by
developing the root-nodules.

The degree of symbiosis in this example is, perhaps, not ab-
solute.! The bacterium can live as an independent organism
in the soil. Its symbiotic qualities are of the nature of partial
adjustment. In other words, the organism has not changed to
such a degree that it cannot exist independently of the host
organism. This status of its existence is undoubtedly respon-
sible for the ease with which the organisms from a root-nodule
may be grown on artificial culture media.

Compared to absolute parasitism, the root-nodule bacteria
are not as dependent on the host as is an absolute parasite. In
the case of an absolute parasite, as well as an absolute symbiont,?
the adjustment is so complete that the organism does not nor-
mally live outside thehost. However, the Bacillusradicicola offers
an illustration of a microscopic organism that may live and
flourish within the cytoplasm of the cells of a higher organism.

1 Various classifications of symbiosis may be found in the literature. The
terms employed in these classifications have been based upon individual examples
and conceptions and consequently can not be employed with clarity in an en-
larged conception of symbiosis. Schneider’s (1897) terms ‘“‘mutualism,” “indi-
vidualism,” and “‘contingent mutualism’”’ are vague and misleading. The terms
‘absolute’ and ‘incomplete’ symbiosis have been introduced by the author on
account of their simplicity, clearness, and direct significance.

2 The author chooses to use the term ‘“‘symbiont’’ employed by Schneider (1897)
in preference to the term ‘‘symbiote’’ introduced by Portier (1918) for the
reason that ‘‘symbiont’’ refers to either one of the two organisms entering into
symbiosis, while ‘“‘symbiote” refers particularly to mitochondria in a bacterial
conception of their nature.

456 IVAN E. WALLIN

Further, it offers evidence of the fact that the chemical products
of the bacterium in this case are essential to the life of the host
plant. True, the host plant may procure these chemicals by
absorption from the soil, and it may even do so in spite of the
nodule organisms. This fact, per se, has no bearing on the im-
portance of the phenomenon. The point at issue may be clarified
by an illustration: The thyreoid gland produces a chemical
substance that is essential to normal metabolism in higher,
animals. The gland may be removed from an animal, but the
chemical substance must be supplied artificially if life is to be
maintained normally. If it were possible to stimulate the pro-
duction of this chemical substance from another organ of the
animal, then the thyreoid would be unessential and in all proba-
bility would degenerate.

Lewitsky (10), Guilliermond (’12), Regaud (11), and other
investigators have ascribed to mitochondria the property of
plastid formation in plants. According to these investigators,
the original mitochondria transform into plastids. Accompany-
ing this transformation the mitochondria take on the various
functions characteristic of plastids. Various kinds of plastids
are to be found in plants. From the standpoint of evolution,
the more important of these plastids are the chloroplasts, or the
plastids containing chlorophyl.

According to Guilliermond, chloroplasts in higher plants are
formed from mitochondria. He has, apparently, observed the
various intermediate stages in the metamorphosis from a minute
body, the mitochondrium, to a fully formed chloroplast. Such
a morphogenesis is so strikingly similar to the morphogenesis
of the Bacillus radicicola in the root-nodules of Leguminosae
that the writer feels justified in presenting his observations on
these forms.

MATERIALS AND METHODS

Root-nodules found on the roots of the common white clover,
growing on the University campus, were fixed in the modified
Flemming’s fixative described in a former paper (Wallin, ’22).
After they were washed, dehydrated, cleared, and embedded

ON THE NATURE OF MITOCHONDRIA 457

in paraffin, they were cut into sections 3y in thickness, mounted,
and stained with Bensley’s aniline fuchsin methyl green.

The author has made no original observations on chloroplast
formation. The work of Lewitsky, Regaud, Guilliermond, and
other investigators in this field is accepted as fully demonstrated.

Bacillus radicicola

A longitudinal section of a root-nodule of the white clover,
when examined with a low magnification of the microscope,
reveals three distinct areas in the nodule. When a higher magni-
fication is employed, the three distinct areas may be seen to be
composed of cells containing three distinct types of bacteria-
like organisms. Figures 1, 2, and 3 are camera-lucida drawings of
portions of these three areas from a single nodule.

In figure 1 the typical Bacillus radicicola may be recognized.
Most of the organisms are rod-shaped. <A few of the characteris-
tic Y-shaped individuals may be seen. It is this type, represented
in figure 1, that, I believe, is generally considered the active in-
dividuals in nitrogen fixation. They may be designated the
‘mature forms.’

In figure 3 the cells contain small bodies that are not unlike
mitochondria in appearance. These forms, undoubtedly, are
the young bacilli that have recently entered the nodule, or
they represent a young form that will later metamorphose into
the mature type. They may be designated the ‘juvenile forms.’

Prazmowski (quoted by Marshall, ’12) and other investigators
have demonstrated that the source of Bacillus radicicola is
from the soil. The free-living forms are quite small. They
enter the roots through the root-hairs. The host plant re-
sponds with the production of the nodules into which the juvenile
forms migrate and later metamorphose into the mature type.
I have not been able to find a comprehensive description of the
metamorphosis.

In figure 2 the organisms found in the cytoplasm of the nodule
cells are spherical in shape. I have been unable to find any
mention of these forms in bacteriological literature. The Ameri-
can text-books on bacteriology have no reference to them.
They may be designated the ‘senile forms.’

458 IVAN E. WALLIN

These senile forms occupy the older part of the nodule. That
is, they are present almost exclusively in the cells of the nodule
nearest to the attachment to the root. Apparently, there is
nothing indicated concerning their activities. Their position
in the nodule suggests the possibility that they are senile. It
is possible that they no longer concern themselves with nitrogen
fixation. However, one is justified in concluding that they are
a stage in the morphogenesis of Bacillus radicicola.

The failure of investigators of this interesting and well-known
organism to notice the existence of the senile forms is, assuredly,
an excusable error. The object of my study, as well as my his-
tological training, suggested the study of the root-nodule cells
intact. The bacteriologist would crush the nodule and make a
smear to study the contained bacteria. Again, the bacteriologist
is interested in the cultural behavior of bacteria. The nodule
must be crushed to transplant the contained organisms.

I have never observed the spherical or senile forms of the
bacillus in smear preparations of the root-nodules.

Figure 4 is a camera-lucida drawing of a portion of the stem of
a clover leaf after mitochondrial fixation and staining. Figure
5 is a camera-lucida drawing of a portion of an unfolded clover
leaf after mitochondrial fixation and staining. These prepara-
tions are introduced to furnish a basis for comparison of apparent
mitochondria in this plant with, particularly, the juvenile forms,
of bacillus radicicola as represented in figure 3. It is apparent
from an examination of these illustrations that the mitochondria
are similar in appearance to the juvenile bacteria. While it is
obvious that form in itself does not necessarily indicate relation-
ship, the question may properly be asked: What evidence is
there to indicate that these structures are cytoplasmic organs
and not representatives of the organisms that are known to be
present in the root-nodules of this plant? Again, it may be asked
with equal pertinence: What evidence may be submitted to
indicate that these bodies, generally considered cytoplasmic
organs, are not bacteria that have gained entrance to the plant
through the root-hairs? The author has not been able to find
any evidence that would satisfactorily answer these questions.

a

ON THE NATURE OF MITOCHONDRIA 459

To recapitulate, thé Bacillus radicicola is a minute organism
that may be found as a free-living bacterium in the soil. Under
favorable conditions, it may enter the root-hairs of Leguminosae
and enter into a partial symbiotic existence. The host plant re-
sponds to the infection with a production of nodule cells. The
invading organism comes to lodge in the cytoplasm of the root-
nodule cells. In a mature root-nodule these forms, apparently,
represent three stages in the morphogenesis of the bacillus after it
acquires the symbiotic relationship.

DISCUSSION

The morphogenesis of Bacillus radicicola from the juvenile
to the senile forms is strikingly suggestive of the morphogenesis
of chloroplasts as described by Guilliermond and other investi-
gators. Both forms develop from minute structures that have
similar form and staining reactions. Both forms have a similar
shape when they have reached their fullest development and are
apparently alike in fragility. Both forms have a similar rela-
tionship to the cytoplasm of host cells. The morphogenesis of
chloroplasts, as described by Guilliermond, appears to imitate
more closely the morphogenesis of an organism than the develop-
ment of a cytoplasmic organ.

The senile forms of Bacillus radicicola have a particular inter-
est and will serve a useful existence in our conception of the nature
of mitochondria. It is quite obvious that absence of these forms
in bacteriological literature is due to the fragility of the organism.
In ordinary bacteriological technique these forms are destroyed;
both in smear preparations and planting on culture media they
would be destroyed in the procedure. I have not used any other
fixation for root-nodules than the one indicated above (a mito-
chondrial fixative). It is probable that the senile forms are
destroyed by many fixatives just as mitochondria are, and this
condition may account for some of the failures of previous in-
vestigators to observe them.

If my interpretation of the senile forms is correct, then, they
furnish excellent proof of the contention I stated in the ‘ad-

460 IVAN E. WALLIN

dendum’ to a former article (Wallin, ’22), namely, that fragility
is a resultant of symbiosis. Further, their fragility refutes
Regaud’s contention that no bacteria are known which exhibit
the fragility of mitochondria.

The apparent fragility of the senile forms of Bacillus radicicola
has a bearing on the problem of growing mitochondria on artifi-
cial culture media. It is apparent from the absence of these
forms in bacteriological literature that they have not been cul-
tivated. This failure to grow them on culture media may be
caused by various factors. It may be possible that they require
a special culture medium. It is more likely, however, that their
fragility does not permit a transfer. A third possibility presents
itself, namely, that they may have lost the power of reproduction.
This latter possibility appears the least likely explanation. It
it quite certain that if success is to attend attempts to grow
mitochondria in artificial culture media, the fragility of mito-
chondria, as well as the proper culture medium, must be taken into
account.

There is no reason to suppose that all mitochondria and sym-
biotic bacteria develop and possess the same degree of fragility.
To the contrary, I have presented evidence in the preceding
section of this paper in support of the contention that mito-
chondria vary in fragility.

It is perhaps excusable at this time to digress into the realm
of theory. The question naturally arises: Would a symbiotic
bacterial interpretation of mitochondria be antagonistic to es-
tablished factors in the theory of evolution?

Osborne (717) has stated a clear and logical conception of
evolution on the earth. His conception includes a chemical
evolution preceding and leading up to the establishment of
definite organisms. The first organisms in this conception were
of a bacterial nature. These primordial bacteria or bacteria-
like organisms were able to subsist on inorganic material. This
conception of the first life is strengthened by the discoveries
of Alfred Fischer (00) and other later investigators. Fischer
discovered a strain of bacteria that apparently represent the per-
sistence of these primordial organisms, the Nitroso monas.

ON THE NATURE OF MITOCHONDRIA 461

The presence of these primordial organisms in the beginning of
evolution established the conditions essential for the develop-
ment of those higher forms that require organic material for
sustenance. It is not difficult to conceive the rdle that the
primordial chlorophyl-bearing organisms played in the modus
operandi of early evolution. Concerning themselves with the
production of starch, the most important of the early food ma-
terials, they undoubtedly assumed the roéle of food factories
preparing the way for the evolution of higher life.

The simplest organisms containing chlorophyl are bacteria
or bacteria-like organisms. These organisms are generally called
the blue-green algae. Campbell (99), Thom (712), Osborne
(17), and other investigators, however, maintain that they
are more closely related to bacteria than to algae. Regardless
of their relationship, whether with the bacteria or with the algae,
the fact remains that chlorophyll is one of the oldest specialized
materials of living matter.

It is in harmony with known biological behavior to conceive
of chloroplasts not as organs developed in the cytoplasm of higher
plants, but to look upon them as bacteria or bacteria-like organ-
isms that accepted the leisure of a symbiotic partnership in the
struggle for existence. In exchange for the nourishment sup-
plied to it by the host plant, the invading symbiont furnishes an
indispensable product to the host organism. It is obvious
that if this interpretation coincides with reality, then the chloro-
plast is an example of absolute symbiosis.

The presence of-chloroplasts in certain animal cells makes
the foregoing hypothesis all the more alluring. It is true, that
in some of these animal cells the chlorophyl-bearing bodies are
unicellular algae that have been ingested by the host organism.
This fact, however, serves to demonstrate the possibility that
certain animal celis are so chemically constituted that algae
may live within their cytoplasm with apparent impunity. The
possibility also suggests itself that unicellular chlorophyl-bearing
organisms may develop a symbiotic relationship with some ani-
mals and that the products of the chlorophyl-bearing organisms
may be essential to the sustenance of the animal cell.

462 IVAN E. WALLIN

In a discussion of algae and their relationships, Campbell
(99) refers to some chlorophyl-bearing algae that have developed
a symbiotic relationship to some higher plants. There is no
reason to suppose that all unicellular green or blue-green algae
belong to a single species. ‘To the contrary, botanists recognize
many species. The fact that some species of algae may be
recognized in a symbiotic relationship with higher plants indicates
the possibility that other species may have developed an absolute
symbiosis with some higher plants. After an organism has de-
veloped absolute symbiosis, it is not conceivable that it is capable
of an independent existence under natural conditions. It is,
also, to be expected that an absolute symbiont would lose some
characters and properties that the free-living progenitor possessed.

One of the best known and oldest examples of symbiosis is
furnished by the lichens. This symbiosis was first described by
Schwendenen in 1860, and has since been confirmed by the in-
vestigations of a host of authors. The lichens furnish not only
an indisputable example of absolute symbiosis, but also varying
degrees of symbiotic relationships.

In the lower lichens the algal symbiont is capable of independ-
ent existence. While it has not been demonstrated, Schneider
(97) and other investigators believe that the fungal symbiont
in some lowly lichen may also be capable of independent exist-
ence. In the most highly developed lichens the symbiosis is
complete or absolute; neither symbiont can live without the
other. Here we have an example of symbiosis that, apparently,
is identical with every detail in the relationship of chloroplast to
host plant. :

Between the most lowly and the most highly developed lichens,
various degrees of symbiosis are represented. This gradation of
symbiosis is accompanied by differences in reproduction. In the
lowly lichens, where symbiosis is incomplete, the algal and fungal
symbionts reproduce independently. In higher forms, but where
the symbiosis is still incomplete, an accessory reproduction may
take place by a type of budding in which both algal and fungal
symbionts are represented. Inthe most highly developed lichens,
where there is absolute symbiosis, both the algal and fungal

ON THE NATURE OF MITOCHONDRIA 463

symbionts enter into the formation of the reproductive organ. This
organ which is comparable to the germ cells of higher organisms
was first named ‘soredium’ by Acharius (from Fiinfstiick, ’07).
Various types of soredia may be found in different lichens. In
general, the fungal symbiont supplies a part (very much of the
character of the spore organ in fungi) which arranges itself around
the algal contribution (the gonidia). In other words, the algal
symbiont is carried from one generation to another in the reproduc-
tive element of the fungal symbiont. ‘This condition is absolute
symbiosis in its highest development.

This latter reproductive phenomenon indicates the solution
to a problem that will assuredly arise in connection with a symbio-
tic bacterial conception of mitochondria: If mitochondria are
symbiotic bacteria, what is their source? Indeed, we would not
seek some avenue of entrance in the adult organism nor in the
embryonic body. The search would logically begin in the germ
cell. The presence of mitochondria in the germ cell has been
fully demonstrated. To seek the original source of a symbiotic-
bacterial mitochondrium would lead us back to the dawn of
evolution. Such an attempt, per se, is impossible. On the
basis of biological behavior as exemplified in the absolute sym-
biotic lichens, there is but one logical answer to the above-stated
question: The symbiotic-bacterial-mitochondria are carried from
one generation to another in the germ cells.

Another question presents itself in connection with a symbiotic
bacterial theory of mitochondria: Does such a theory harmonize
with the known factors of cell activity? Altmann (’90) originally
advanced the theory that the ‘bioblasts’ (mitochondria) are
the ultimate units of life and the cytoplasm of the cells in which
they are contained is lifeless. Altmann’s ideas, apparently, were
chiefly theoretical. Verworn (’99) and other investigators dem-
onstrated the untenable position of suchahypothesis. Abundant
evidence may be offered to prove that cytoplasm, by itself,
possesses properties that are characteristic of living matter.
{f Altmann had limited his dissertation to a consideration of
the ‘bioblasts’ alone, the burden of proof would have fallen on his
adversaries. He misled his critics by introducing an erroneous
conception of cytoplasm. This brought forth ridicule from his

464. IVAN E. WALLIN

critics instead of further scientific analysis. Following Altmann’s
dissertation, a great amount of theory has been advanced con-
cerning mitochondria and cytoplasm. If we analyze many of
these theories we find very little if any real evidence as a basis for
their pronouncement.

The cell may be considered a unit chemical factory into which
materials are almost continually passing and products are being
eliminated. The simplest type of cell, if it may be considered
a cell, is the bacterial organism. It is simple in the sense that it
responds more readily to environmental conditions than do the
highly differentiated cells of complex organisms. Gourney-
Dixon (’20) has collected a mass of evidence in this direction.
During recent years there has been a great deal of evidence
submitted to show that the cells of one tissue are dependent upon
the product of the cells of other tissues. Adami (’10) even goes
so far as to state that every cell in a complex organism performs
the function of internal secretion. Such a relationship of cells
is also present among the lowly bacteria and is designated as
symbiosis by bacteriologists. This principle of cell dependence
is, thus, not characteristic of the cells of higher organisms, but
is a primitive adjustment. Further, it is highly probable that
this principle of cell dependence has been a fundamental factor
in organic evolution.

The symbiotic bacterial conception of mitochondria, ap-
parently, is not antagonistic to any known principles of cell
activity. On the other hand, such a conception only extends
the ‘cell dependence principle’ to include an intimate dependence
of all highly organized cells on the activities of simple cells. The
Bacillus radicicola in the root-nodules of Leguminosae is incon-
trovertible testimony that such a functional and morphological
relationship does exist.

The dependence of higher life on bacteria in a different sense
has been embodied into the biological conceptions of all students
in biology. The principle of the ‘nitrogen cycle’ stands sponsor
to these conceptions. It is within the domain of logic to extend
this well-established principle of dependence of higher life on
bacteria to include the more intimate dependence of life processes
on bacteria.

ON THE NATURE OF MITOCHONDRIA 465

GENERAL RESUME

From the evidence that has been submitted in this study, in-
cluding sections I and II in a former paper, the author believes
that he has demonstrated the following facts:

There is no fundamental difference in the staining reactions of
mitochondria and bacteria.

There is no fundamental difference in the reactions of bacteria
and mitochondria to certain chemicals that have been used in
attempting to determine the chemical nature of mitochondria.

Mitochondria may be demonstrated by bacteriological
methods.

Bacteria may be demonstrated by mitochondrial methods.

Mitochondria vary in fragility.

Bacteria vary in fragility.

Mitochondrial substance is apparently miscible with the
cytoplasm of the host cell.

The author, further, believes that the following apparent
facts and biological principles support a bacterial nature of
mitochondria:

Similarity of form.

Similarity in staining reaction.

Similarity in chemical ractions.

Similarity in physical properties (fragility).

Similarity in functional properties (synthesis).

In harmony with principle of biological behavior as exempli-
fied in the ‘struggle for existence’ resulting in symbiosis.

In harmony with known factors of evolution.

In harmony with known factors of cell activity.

In harmony with ‘principle of cell dependence.’

In harmony with the principle of analogy (Bacillus radicicola,
lichens).

466 IVAN E. WALLIN

CONCLUSIONS

In a ‘balance sheet’ of favorable and unfavorable evidence
in behalf of a bacterial nature of mitochondria, it appears to the
author that the ‘unfavorable’ side of the ‘sheet’ lacks entries.
After careful analysis, the author is convinced that no property
of mitochondria has been recorded in the literature that is not
equally applicable to bacteria.

From the evidence that has been recorded in these studies,
together with the evidence that may be found in mitochondrial
literature, the author can arrive at no other conclusion than, that
mitochondria are symbiotic bacteria in the cytoplasm of the cells of
all higher organisms whose symbiotic existence had its inception
at the dawn of phylogenetic evolution. The conception embodied
in this conclusion presupposes that the establishment of new
symbiotic complexes is coexistent with the development of new
species.

These studies have been pursued by the author independently.
The conceptions and principles stated in this article have been
formulated entirely on the basis of evidence that the author has
gathered. Portier (’18) has arrived at a similar conception of the
nature of mitochondria. His treatise ‘Les Symbiotes’ has not as
yet been read by the author. A critical analysis of Portier’s
book will be undertaken by the author in the near future.’

3 After this article had been received by the publishers I found a reference to
the spherical forms of Bacillus radicicola. Léhnis (1921 Studies upon the life
cycles of bacteria. Pt. 1. Memoirs of the National Academy of Sciences,
Vol. XVI, 2nd memoir. Wash.) has described spherical forms of this organism.
The spherical forms are of different sizes and, according to Léhnis, represent
morphogenic stages in the life cycle of Bacillus radicicola.

ON THE NATURE OF MITOCHONDRIA 467

LITERATURE CITED

ApamtI, GrorGe J. 1910 Principles of pathology. Philadelphia and New York.

ALTMANN, R. 1890 Die Elementaroraganismen und ihre Beziehungen zu den
Zellen. Leipzig.

CaMPBELL, D. H. 1899 Lectures on the evolution of plants. MacMillan,
New York.

Cowpry, E. V. 1918 The mitochondrial constituents of protoplasm. Carnegie
Inst. Bub., Contr. to Embr., no. 25, Washington.

FiscHER, ALFRED 1900 Quoted by Osborn (1917).

Finrstiicx, M. 1907 Article on lichens in Die natiirlichen Pflanzenfamilien,
I. Weil, I. Abt. Leipzig.

GuRNEY-D1xon 1920 The transmutation of bacteria. Cambridge.

GUILLIERMOND, A. 1912 Recherches cytologiques sur le mode de formation de
V’amidon et sur les plastes végétaux, ete. Arch. d’anat. micr., T. 14,

Lewitsky, G. 1910 Ueber die Chondriosomen in pflanzlichen Zellen. Ber. d.
deutsch. bot. Ges., Bd. 28.

MarsuHatn, C.K. 1912 Microbiology. Philadelphia.

Osporn, Henry Farrrretp 1917 The origin and evolution of life. New York.

Reeaup, Cu. 1911 Les mitochondries. Rev. de Méd., T.31.

ScHWENDENER 1860 Quoted by Fiinfstiick (1907).

THom 1912 In Marshall (1912).

Topp, JAMEs C. 1918 Clinical diagnosis. Philadelphia.

Verworn, Max 1899 General physiology, an outline of the science of life.
Translated by Lee. New York.

Wattin, Ivan E. 1922 On the nature of mitochondria. I. Observations on
mitochondria staining methods applied to bacteria. II. Reactions of
bacteria to chemical treatment. Am. Jour. Anat., vol. 30, no. 2.

EXPLANATION OF PLATES

The figures were made with the aid of the camera lucida. The lenses used
were: 2-mm. apoch. oil.-immer. obj., comp. ocular no. 8. The figures in plate 1
are reduced one-half from the original. The figures in plate 2 are reduced only
very little from the original.

PLATE 1
EXPLANATION OF FIGURES

1 Mature forms of Bacillus radicicola in the cytoplasm of root-nodule cells.
Bensley’s stain.

2 Senile forms of Bacillus radicicola in the cytoplasm of root-nodule cells.
Bensley’s stain.

3 Juvenile forms of Bacillus radicicola in the cytoplasm of root-nodule cells.
Bensley’s stain.

4 Mitochondria in the leaf stem of the white clover. Bensley’s stain.

5 Mitochondria in a young unfolded leaf of the white clover. Bensley’s stain.

6 Rabbit pancreas smear after pyronin-methyl green staining.

7 Rabbit liver smear after pyronin-methyl-green staining.

468

ON THE NATURE OF MITOCHONDRIA PLATE 1
IVAN E. WALLIN

469

PLATE 2
EXPLANATION OF FIGURES

8 Rabbit kidney smear after pyronin-methyl-green staining.
9 Rabbit lymph-node smear after pyronin-methyl-green staining.

470

PLATE 2

ON THE NATURE OF MITOCHONDRIA

&. WALLIN

IVAN

471

tesumen por el autor, A. W. Bellamy.

La suseceptibilidad diferencial como base para la modificacién y
regulacion del desarrollo de la rana.

II. Tipos de modificacién observados en estados mids tardfos
del desarrollo.

I. El 6vulo y el embrién de la rana exhiben una susceptibilidad
diferencial a una gran varidad de condiciones que trastornan
los procesos del desarrollo. 2. Segtin el tratamiento experi-
mental y la condici6n fisiol6gica del organismo en el momento
de la exposicién, las modificaciones inducidas caen naturalmente
dentro ce clases de inhibicién diferencial, aceleraciones difer-
enciales, aclimataciones diferenciales o vueltas a la normalidad
diferenciales. 3. Estas modificaciones diferenciales son conse-
cuencias obvias y necesarias de la susceptibilidad diferencial
del organismo a los agentes empleados en la teratologia experi-
mental. 4. La respuesta de desarrollo del 6vulo y embrién de
la rana a una variedad de agentes externos es primariamente
cuantitativa y no especifica. 5. Los gradientes en suscepti-
bilidad son paralelos a los ejes de simetria. Las regiones que se
diferencian mds temprano y crecen mas rapidamente son las
que experimentan mayor desplazamiento bajo las condiciones que
trastornan los procesos del desarrollo. El grado y direcci6én
del desplazamiento dependen en su mayor parte de la severidad
del tratamiento y la condici6n fisiol6gica del organismo. 6. La
existencia de gradientes de susceptibilidad, demostrada en una
eran variedad de organismos, necesariamente implica la exis-
tencia de un gradiente o gradientes en los aspectos estructurales
y funcionales del protoplasma en general. 7. El autor cree que
el desarrollo teratolégico puede explicarse mds racionalmente en
términos de este 6rden graduado de las relaciones dindmicas y
actividades del protoplasma: los gradientes fisiolégicos.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MAY l

DIFFERENTIAL SUSCEPTIBILITY AS A BASIS FOR
MODIFICATION AND CONTROL OF DE-
VELOPMENT IN THE FROG

II. TYPES OF MODIFICATION SEEN IN LATER DEVELOPMENTAL
STAGES

A.W. BELLAMY
Hull Zoological Laboratory, The University of Chicago

SEVENTY FIGURES
INTRODUCTION

The fact that it is possible, within limits, to modify and con-
trol the developmental processes of a wide variety of organisms
lends weight to the conception that the organism represents a
complex or ‘pattern’ which, while it is characteristic of the species,
entails a variety of ultimate ontogenetic possibilities. This or
that aspect of development is revealed according as the develop-
ing organisms is exposed to this or that constellation of environ-
mental factors. Viewed in this light, teratological development
is simply a consequence of a deviation from the usual trend of
development. Now, development may be made to depart from
its usual trend in a variety of ways, and the degree or extent
of the deviation from the usual course of development seems to be
limited, for the most part, by two general factors. ‘The primary
limiting factor is of course the nature of the ‘organismic pattern,’
as Child (’20, p. 148) termed it. Since a wide variety of environ-
mental factors has been used successfully to produce essentially
similar abnormalities or deviations from the ‘normal,’ the second
limiting factor would seem to be the intensity! of action of these
agents rather than any specific action that might be ascribed to
them.

1 The word ‘intensity’ is used here in the sense of, e.g., concentration with
reference to a chemical substance in solution. ;

473

THE AMERICAN JOURNAL OF ANATOMY, VOL. 30, NO. 4

474 A. W. BELLAMY

Teratological developments certainly have a number of fea-
tures in common regardless of how or in what organisms these are
induced: One of these features is differential susceptibility, a
differential that bears a very definite relation to the ‘physiological
axes’ of the organism. This means simply that different regions
of the physiological axes of organisms show different degrees of
susceptibility to certain concentrations of action of a variety of
physical and chemical agents. Observations by Child and _ his
students on some two hundred. species of organisms, including
representatives from all the principal animal phyla and a con-
siderable number of plants, agree in showing, at least in the
simpler organisms and in the early stages of development of
higher forms, that those regions of the egg or embryo that dif-
ferentiate earliest and grow most rapidly, or simply the most
active regions of the organism, are most susceptible to conditions
that seriously affect developmental or functional processes.
Several methods are available for demonstrating these differences
in susceptibility,? the differential appearing, according to experi-
mental conditions, as a differential disintegration gradient
associated with death, as differential inhibition, as differen-
tial acceleration, or as differential acclimation or recovery in
development.

The fact that such a wide variety of organisms, both plant and
animal, characteristically exhibits a differential susceptibility to
a considerable number of external agents and conditions (cya-
nides, anesthetics, acids, alkalies, certain electrolytes, several
alkaloids, extremes of temperature, lack of oxygen, et al.) indicates
at once that there is something fundamentally similar in the _
organismic pattern existing in these different protoplasms. It
must mean that organismic pattern, as distinguished from the

2 Differences in susceptibility along the axes of organisms are demonstrable
as: 1) differences in survival time of one region of the organism as compared
with other regions, under conditions that kill slowly without permitting acclimation
to occur; 2) as differences in the degree of inhibition of growth and development,
or in certain cases as differences in the degree of acceleration of these processes;
3) as differences in the rate or degree of acclimation to a certain range of less
severe conditions; 4) as differences in the rate or degree of recovery after tem-
porary exposures that inhibit development. See also Child, ’20, p. 154.

MODIFICATION OF DEVELOPMENT IN THE FROG 475

specific protoplasmic constitution of each particular type or
species, represents in at least certain fundamental respects a
non-specific or quantitative factor in organization characteristic
of at least all axiate organisms and transcending the differences
in details which constitute specific or type differences in
organisms. ‘To this fundamental feature of organismic pat-
tern, Child (20, p. 152) has applied the descriptive term, ‘physio-
logical gradient.’ Differential susceptibility or, in other words,
a gradient in susceptibility, then, is merely one expression of
underlying graded differences in the rate of fundamental activities
and dynamic relations in protoplasm. A summary and discus-
sion of the evidence demonstrating the existence of physiological
eradients in organisms is given in the above citation so that
further discussion of this principle is unnecessary. I wish merely
here to indicate the usefulness of the conception in the interpre-
tation of teratological development. Indeed, since the various
types of experimentally produced terata in the frog have been
produced time and again through the action of almost every
agent and device known to experimental teratology, there would
be little excuse for going over the ground again except for two
facts which now appear very clearly. First, the degree and di-
rection of teratological modification of development evidently
depend primarily upon quantitative rather than specific factors
in the action of the agents used; and, second, on this basis, the
data attain considerable added significance and can be so satis-
factorily accounted for in terms of the conception mentioned
above.

In 1919 I presented data to show: 1) that the frog egg and
embryo exhibit a differential susceptibility to such agents as
KNC, CH.O, KMnO,, LiCl, HCl, NaOH, and C.H;OH. Those
regions of the egg which differentiate earliest and in which growth
is most rapid (apical and dorsal regions—in the early stages of
development; in later stages any rapidly proliferating or physio-
logically active region) die soonest in concentrations of external
agents that are lethal within a relatively short time; are most
inhibited by somewhat lower concentrations, and acclimate or
recover first in concentrations or treatments permitting acclima-

476 A. W. BELLAMY

tion and recovery. 2) In general it was found that any type of
abnormality could be produced by, a) regulating the concentra-
tion or intensity of action of a given agent; b) regulating the
length of exposure of the egg to the agent—in connection with a
and c; c) varying the stage in development at which the egg or
embryo is exposed to the experimental conditions. 3) It was
further emphasized that, fundamentally, the frog egg and em-
bryo reacted to the agent or conditions used not specifically,
but quantitatively. In other words, the end result is primarily
a function of the intensity of action of the agent or condition
used, the length of exposure, and the physiological condition of
the eggs at the time of exposure to the experimental conditions.

The previous report dealt primarily with experimentally pro-
duced modifications seen in the earlier stages of development—up
to and including gastrulation. This report is concerned for the
most part with the modifications seen in the later stages of de-
velopment—gastrulation to the time of hatching or a little later.

I am under obligation to Prof. C. M. Child, in whose laboratory
the work was begun in 1916, for aid in the way of suggestion and
criticism. .

To facilitate the presentation of the experimental data and to
give a brief sketch of the results of the experiments, the charac-
teristic features of the different types of modified development are
described below, together with a statement of the general experi-
mental conditions under which the different types of modifica-
tions may be expected to appear.

TYPES OF MODIFIED DEVELOPMENT

In spite of the almost endless variety of abnormal forms that
appear in experiments of this nature, the types of abnormalities
obtained fall readily into four general categories, according as
development has been inhibited or accelerated, or according as
the egg or embryo acclimates to the experimental conditions, or,
on removal to water, recovers. Furthermore, however different
in appearance embryos of these different types may be, there is
at least one characteristic feature common to all of them. The
response of the egg or embryo is differential, whether the condi-

MODIFICATION OF DEVELOPMENT IN THE FROG A477

tions be inhibiting, accelerating, or such that the embryo accli-
mates to them, or, on removal to normal conditions, recovers.
Hence one finds the distortion in development, regardless of the
type, whether inhibition, acceleration, acclimation or recovery,
appearing in relation to the principal axes or planes of symmetry
of early developmental stages—anteroposterior, dorsoventral, and
mediolateral—and to the secondary axes of special organs arising
later in development. In early development before regional
differentiation has begun, anterior, dorsal, and medial regions
which are physiologically more active than their opposites, are
more affected by disturbing conditions of the four sorts men-
tioned above than the physiologically less active posterior, ven- _
tral, and lateral regions. This is the typical condition. With
the origin of special structures, however, optic vesicles, posterior
growing region, limb buds, e.g., or in other words, with the origin
of the secondary physiological gradients of special organs, the
typical condition may be considerably modified, at least tempora-
rily. Such special structures, which, it may be mentioned in
passing, arise in an orderly relation to the primary axes, May
‘flare up’ with a high initial rate of metabolism that may persist
through the period of most rapid proliferation and differentiation.
The optic vesicles, e.g., for a time represent the most susceptible
region of the anterior end of the embryo. | Similarly, the tail
bud, which terminates the posterior growing region, is at the time
of its origin quite as or may be even more susceptible to disturbing
conditions than more apical regions. These constantly changing
relations in the developing embryo between the primary physio-
logical gradients and the gradients of special organs arising later
in development, together with the individual variation in the
physiological condition of the embryo as a whole, make absolute
uniformity among the modified embryos, whether inhibitions,
accelerations, acclimations, or recoveries, highly improbable, to
say the least. Great variation is to be expected in every case.
The four types of modifications obtained are characterized as:
differential inhibitions, differential accelerations, differential ac-
climations, and differential recoveries. They are described in the
order mentioned. It is hardly practicable or even desirable here

Figs.1 to9 Differential inhibition forms. 1, lateral; 2, dorsal view of one
embryo. Eggs in late cleavage stages when placed in the solution. Treatment:
53 hours in m/10 LiCl. Note the elongated yolk plug which appears to result
primarily from the relatively greater retardation of the dorsal lip region. Two
outline views of the same embryo made sixteen and one-half hours later are
shown in figures 52,53. 3, same as above, but after 93 hours in m/10 LiCl. Ven-
tral suckers, v.s., are ‘fused.’ 4, 5, 6, three embryos from same lot as the one
shown in 3. 7, lateral, 8, dorsal view of one embryo, and 9, lateral view of a
second embryo. Eggs unsegmented when placed in the solution. Treatment:
76 hours in m/10.62 LiCl + 20 hours in water. 6, open brain region; y.p., yolk
plug; v.s., ventral sucker. The anterior end is toward the top of the page in all
figures, save 3. Figures 1 to 6, from Exp. IV 59; 7 to 9, from Exp. IV 22; 4, 5, 6,

drawn to slightly smaller scale.
478

a

MODIFICATION OF DEVELOPMENT IN THE FROG A479

to attempt any detailed description of the great variety of forms
belonging to any of the four types. Hence, in the following four
sections only the characteristic general features of the types will
be pointed out. The conditions under which the different types
occur may be seen from the legends to the figures and by reference
to the protocols of experiments.

Differential inhibition. The modified types that fall into this
class, while highly variable, have the following common charac-
teristics: Physiologically more active regions suffer relatively
greater retardation than less active regions. Among the more
conspicuous external features of differential inhibition forms seen
in postgastrula stages are these: Embryos with an elongated
persistent yolk plug and retarded apical region (figs. 1, 2), or
with a very marked retardation of apical dorsal parts and showing
an equatorial blastopore with a persistent yolk plug (figs. 7, 8,
9). The later type grades back, in more completely inhibited
forms, to a condition where apical differentiation does not pro-
ceed and the embryo dies as an equatorial gastrula. Where de-
velopment proceeds further from such a condition, the embryo
appears to elongate in the direction of the old egg axis, resulting
in types that may appear to be radially symmetrical or may pro-
ceed to the formation of embryos similar to those shown in figures
3, 4, 5, 6. In somewhat later stages one characteristically finds
microcephalic or even anencephalic embryos with such structures
as optic vesicle, olfactory pits, and ventral suckers, when they
appear at all, in various degrees of approximation to complete
‘fusion’ (figs. 10 to 32). In the absence of suitable conditions
or opportunity for recovery, such embryos are usually dorsally
concave, with the tail in some cases extending dorsalward at
an angle of 90° or less with the body (fig. 22). The yolk plug
may or may not be persistent, but the blastopore always, when
it closes at all, does so relatively much later than in normal em-
bryos. Spina bifida forms are of common occurrence (figs. 23, 26).

These various types are easily referable to the relatively greater
retardation of physiologically more active regions, such as apical,
medial, and dorsal regions, and later, the blastopore lips, pri-
mordia of optic vesicle, olfactory pits, ventral suckers, etc.

480 A. W. BELLAMY

22

P-yp 25

Figs. 10 to26 Differential inhibition forms. 10, lateral and, 11, dorsal view
of one embryo. Eggs in two-cell stage when placed in the solution. Treatment:
4 days in KNC + 2 days in water. 12, 13, dorsal views of two embryos. Eggs
four to eight-cell stages when placed in the solution. Treatment: 48 hours
in 0.0075 per cent formaldehyde. 14, ventral view of an embryo from the same
lot as 12, 13, but after 72 hours in 0.0075 per cent formaldehyde. This figure
illustrates a typical spina bifida condition with the two tails projecting dorsally
from cither side of the yolk plug. 15 to 21, eggs early gastrula stages when

us

MODIFICATION OF DEVELOPMENT IN THE FROG A481

Differential acceleration. Those types of deviation from the
usual trend of development that are characterized. as differential
acceleration involve the same regions that are distorted in in-
hibited development, but the changes are opposite in direction.
Regions of the egg, normally most active, become, under condi-
tions that markedly accelerate development, even more preco-
cious. As seen in later development, the conspicuous feature of
accelerated development is a megacephalic condition of the em-
bryo in which the gill plates and ventral suckers are unusually
prominent. There is also a tendency toward a dorsal convexity.

It may be remarked in passing that differential acceleration
forms do not deviate from the usual trend of development to
anything like the extent seen in inhibited development—which,
from the nature of the case, could hardly be otherwise.

Differential acceleration differs from acclimation and recovery
in that the embryos develop more rapidly than the controls—a
circumstance that is especially noticeable during the early cleay-
age stages when the susceptibility is still relatively low. Not
infrequently eggs may be accelerated during early cleavage stages
and severely inhibited or killed in the same solution later in de-
velopment. In mercuric chloride, e.g., eggs may be considerably
accelerated during the first two or three cleavages in m,/10,000
and killed during gastrula stages in m/2,000,000.

Acceleration is seen to a greater or lesser extent in low concen-
trations of all of the substances used, but no special efforts were
made to determine the exact conditions for its appearance with

placed in the solution. Treatment: 12 hours in HCl + 81 hours in water. The
‘fused’ suckers can be seen as cone-like structures protruding from the anterior
ventral region. 22, lateral view of an embryo, to show the marked dorsal con-
cavity frequently seen in differential inhibition forms. Olfactory pits, ventral
suckers, and probably optic vesicles ‘fused.’ Eggs late cleavage when placed in
the solution. Treatment: 3 hours in m/5 LiCl + 8 days in water. 23, lateral
view of spina bifida embryo. Eggs two-cell stage when placed in the solution.
Treatment: 4 days in m/10,000 KNC + 8 days in water. 24 to 26, eggs in early
gastrula stages when placed in the solution. Treatment: 3 hours in n/300 NaOH
+ 6 days in water. Ventral suckers and nasal pits ‘fused.’ 26, dorsal view of a
spina bifida form with a persistent yolk plug. ¢, tail; y.p., yolk plug; v.s., ventral
sucker. 10, 11, 23, from Exp. KNC-C. 9; 12 to 14, from Exp. IV 62;15 to21, from
Exp. 103.3; 22, from Exp. IV 58; 24 to 26, from Exp. 125.2.

A82 A. W. BELLAMY

any substances save the ones mentioned in connection with the
figures which illustrate this type of modification—figures 27 to
38.

Differential acclimation and differential recovery. Both aecli-
mation, which oecurs while the egg or embryo is still exposed to
the experimental conditions, and recovery, which occurs after
the egg or embryo is removed to water, involve the same regions
of the developing organism that suffer distortion in inhibition or
acceleration. Both are differential. These two conditions are
to be distinguished from acceleration in that embryos produced
under conditions that accelerate development are always in ad-
vanee of the controls, while the differential acclimations and

Figs. 27 to 29 Differential acceleration. 27, control; 28, embryo same age as
control after 8 days’ continuous exposure to n/500 NaOH (solution not changed
after third day) from an early gastrula stage. Exp. NaOH 2. 29, embryo after
96 hours’ exposure to m/100,000 KNC. Eggs unsegmented when placed in the
solution. Hxrp. KNC-C.4. v.s., ventral suckers.

recoveries are usually smaller than controls and differentiation
has not proceeded as far. Indeed embryos that show acclimation
or recovery or both may at the same time show a differential in-
hibition due to an earlier treatment. This is more especially
true of the differential recovery forms.

As regards acclimation of the egg or embryo in the various solu-
tions used, my data are not complete. Such data as have been
obtained are brought out incidentally in connection with other
experiments. A number of experiments, however, furnish con-
ditions where recovery might be expected to take place (pp. 486,
491).

Among the more conspicuous features which mark recovery
forms differentially are: relatively large tail buds on otherwise —
differentially inhibited embryos and a marked dorsal convexity
is characteristic.

MODIFICATION OF DEVELOPMENT IN THE FROG 483

37

Figs. 30 to 38 Differential acceleration. n/5000 HCl. Eggs two to four-cell
stages when placed in the solution. 30, embryo after 60 hours in HCl, dorsal
view; 31, controlat60 hours. 32, ventral and, 33, lateral view of one embryo after
96 hours in HCl; 34, lateral view of control at 96 hours. 37, lateral and, 38,
ventral view of head of one embryo after 6 days in HCl; 35, control, lateral view
and, 36, ventral view of head, at6 days. a, anterior; m, mouth; md., medullary
plate; 0, olfactory pit; v.s., ventral suckers; y.p., yolk plug. 30 to 35, Exp. HCl-
C'.1; 35-38, Hzp. HCI-C.5.

484 A. W. BELLAMY

The conditions under which recovery will occur to a suff.cient
degree to reveal a marked differential are necessarily some-
what exacting. The strength of the solution (if a chemical sub-
stance is used), the length of exposure, and the physiological
condition of the embryo must all be taken into consideration.
If the conditions are too severe, recovery will not take place at
all. Furthermore, the relation between different regions of the
physiological axes of the developing organism, as regards rate or
degree of activity, is continually changing. During early cleav-
age stages the apical pole represents the region of greatest
activity as measured by rate of cell division, concentration of
protoplasm, etec., and is most susceptible to a variety of external
agents and conditions. Later the dorsal lip of the blastopore
arises, probably by physiological isolation,? as a secondary
region of high susceptibility.‘ In fact, this secondary or posterior
growing region is at the time of its appearance and for some time
afterward quite as or even more susceptible than the apical region.
Apparently it represents during this time the most actively grow-
ing region of the embryo. Hence the physiological condition of
the embryo at the time of exposure to the inhibiting conditions
and at the time of removal from them, as well.as the severity of
the conditions, must play an important part in determining the
rate and extent of development of those regions emphasized in
recovery after removal from the inhibiting conditions.

It is usually a great satisfaction to the reader to have before
him the complete records of experiments of this nature, but since
the chief purpose of this report is to direct attention to the
fundamental similarity of teratological forms however produced
and to offer a physiological interpretation of the data, and es-
pecially since many of the essential facts of abnormal develop-
ment in the frog have been recorded by a considerable number of

3 See Bellamy, 719, pp. 349, 350.

4The at least temporary independence of the dorsal lip region is further
indicated by certain of Spemann’s (718) recently published experiments. Taking
two triton eggs at the beginning of gastrulation, he removed and exchanged
between the two eggs the entire animal pole cap of cells, at the same time rotating
them through 90°. In these cases he found the anterior end of the medullary
plate developing in apposition to the posterior end of the lower half of the egg.

MODIFICATION OF DEVELOPMENT IN THE FROG A85

investigators, it has seemed unnecessary to burden the reader
with a detailed account of all of the experiments done in this
laboratory. That the results are consistent and repeatable is
apparent at once from the literature of the subject. I have
carried out some two hundred series of experiments, involving
at least one repetition for each experiment. Those involving
lithium chloride, potassium cyanide, mercuric chloride, hydro-
chloric acid, sodium hydrate, and formaldehyde were. repeated
anywhere from three to six or eight times. The experiments
described are given usually in protocol form and involve mostly
agents not hitherto used in experimental teratology (frog).
Several experiments with LiCl are described in some detail be-
cause of the attention that, in the past, has been directed to it as
having a ‘specific’ effect in the production of abnormal forms.
It should be mentioned that of the four types of modified de-
velopment recognized here, three, viz., differential inhibition,
acclimation, and recovery, are more or less the same thing that
Stockard (21) refers to in Fundulus as ‘developmental arrests,’
or, as we would put it, differential developmental arrests. Accli-
mations and recoveries are of course primarily differential in-
hibition forms that have, under a change in the experimental
conditions undergone a remodification that is opposite in direc-
tion to the previous inhibition. The type characterized here as
differential accleration is in addition to the one general type of
modified development recognized by Stockard for Fundulus.

EXPERIMENTAL DATA

Experiments with lithium chloride. Eggs unsegmented at the begin-
ning of the experiment. inthis series the concentrations of LiCl used
were: m/7 (experiment IV 20); m/8.5 (experiment IV 21), and m/10.62
(experiment IV 22).° These concentrations in percentages are approxi-
mately, 0.6, 0.5, and 0.4, respectively. Eggs from a single female,
one and one-half hours after deposition, were divided into four lots,
5 ec. of egg mass to each. Three lots were placed, each into a liter of
the above solutions, the fourth constituting the control. The time at
which eggs were examined is measured from the time the eggs were
placed in the solutions. The temperature where the experiments
were carried on was maintained at 17 + 1°C.

5 Certain phases of these experiments were discussed in the previous report
(Bellamy, ’19, pp. 339-340). Through a typographical error, experiment IV 20,
middle page 339, appears as IV 21.

486 A. W. BELLAMY

In m/7 LiCl development comes to a standstill within about six
hours after the eggs are exposed to the solution, although they are not
killed outright, as shown by the fact that even after twenty-one hours’
exposure development proceeds somewhat further when the eggs are
returned to water. In m/8.5 LiCl eggs, when placed in the solution
before segmentation begins, do not develop much beyond late cleavage
stages unless returned to water, when they go ahead to form equatorial
gastrulae.

Of the differentially inhibited forms produced in m/10.62 LiCl,
those of especial interest here are the ones seen after forty eight hours’
exposure to the solution plus twenty hours in water; forty-eight hours
in LiCl plus forty-eight hours in water, and those exposed seventy-six
hours to LiCl then removed to water for twenty hours or more. Fig-
ures 39 to 46; 7 to 9, will show more clearly than any amount of de-
scription the differential nature of the inhibition suffered under this
treatment. Figures 39, 40 illustrate two embryos after forty-eight
hours’ exposure to m/10.62 LiCl plus the subsequent twenty-eight
hours in water. Figure 39 represents a dorsal view showing a perma-
nent yolk plug, shifted somewhat asymmetrically toward the right;
small head region, with the neural groove still open anteriorly. Figure
AO is a lateral view of another embryo from the same lot as the above.
Figures 41, 42 represent two embryos from the same lot as the above
after seventy-three hours in water following a previous exposure of
forty-eight hours to m/10.62 LiCl. The embryo 41 presents a perma-
nent yolk plug projecting dorsally and showing practically no recovery
of the posterior growing region. The head region is relatively small,
but the ventral suckers are only slightly reduced. The embryo shown
in 42 is markedly microcephalic, with no trace of the ventral suckers,
the yolk plug projecting more ventrally and showing a considerable
recovery of the posterior growing region (tail bud). While the majority
of embryos from this experiment approximated the type shown in
figure 42, there were a number similar to the others illustrated in figures
39 to 41, as well as practically all intermediate stages between the two
types. These variations in the degree and type of recovery under these
severe inhibiting conditions represent in all probability simply individual
differences in susceptibility. Furthermore, the concentration used is
of such severity that it lies near the border-line between conditions
where recovery will not take place at all and conditions where it occurs
readily. Consequently, considerable variation is to be expected (p. 484).

Figures 43 to 46 illustrate three embryos from experiment IV 22a.
The treatment was: forty-eight hours in m/10.62 LiCl and subsequently
eighty-three hours in water. Figures 45 and 46 are lateral and ven-
tral views, respectively, of the same embryo. All of the embryos are
microcephalic, ventral suckers partially or completely ‘fused,’ and with
yolk plugs still protruding. A considerable number of forms with
spina bifida appear. Differential recovery is evident in a number of
them (fig. 44), as evidenced by the dorsal convexity and relatively
large tail buds.

MODIFICATION OF DEVELOPMENT IN THE FROG 487

Figures 7 to 9 illustrate two embryos from the same lot of eggs, but
exposed seventy-six hours to the solution and returned to water for
twenty hours. Differential inhibition is more marked. Many of the
embryos approach an anencephalic condition, and the bilateral sense
organs of the head are much reduced or are not apparent at all. The

Figs. 39 to 46 Differential inhibition. 39, dorsal, 40, 41, 42, lateral views ot
four embryos after exposure to m/10.62 LiCl. Eggs unsegmented when placed
in the solution. Treatment: 39, 40, 48 hours in LiCl + 28 hours in water; 41, 42,
48 hours in LiCl + 73 hours in water. Exp. IV 22. y.p., yolk plug; t, tail; v.s.,
ventral sucker. 43, 44, lateral views of two embryos and, 45, lateral, 46, ventral
view of a spina bifida embryo from same experiment as those shown in figures 39
to 42, but after an exposure of 48 hours in LiCl + 83 hours in water. The em-
bryos 43 to 46 show some differential recovery.

A488 A. W. BELLAMY

neural groove rarely closes and the posterior growing region is almost
completely inhibited. After this treatment usually not more than
half of the eggs continue development at all after return to water. In
a number of embryos no external indications of bilaterality or dorso-
ventrality were apparent. The embryos approximated a radial type
of symmetry. ‘Differentiation’ proceeded from an equatorial gastrula
condition, merely as an apical proliferation of the pigmented cells
and an elongation, as nearly as one could judge, in the direction of the
original polar axis. One of these embryos was illustrated in the pre-
vious report (fig. 20).
Lithium chloride

Eggs in late cleavage stages at the beginning of the experiment
(experiments IV 58 and IV 59). Experiment. IV 58. Eggs in late
cleavage stages (26 hours after deposition) were placed in m/5 LiCl.
At intervals of 14, 3, 5, and 18 hours, eggs were removed to water.

Lot A. (a) 9 hours in LiCl. None of the eges have begun gastrula-
tion. Control: early gastrula.

(b) 18 hours in LiCl. Not much advance. About 5 per
cent show slight wrinkling about midway between equatorial region
and the apical pole. Many dead or dying. Control in late gastrula
and early yolk plug stages.

Lot B. (a) 14 hours in LiCl plus 73 fone in water. Some of the
eggs show slight. “indications of Pale onal gastrulation (deep wrinkling
in equatorial region of the egg).

(b) 14 hours in LiCl, plus 51 hours in water. Embryos
beginning to plgncane. Behind control.

Lot C. (a) 3 hours in LiCl, plus 6 hours in water. The eggs are in
approximately the same condition as those in B (a).

(b) 3 hours in LiCl, plus 49} hours in water. All of the
eggs show marked differential inhibition. Neural fold stages with
large and (probably) permanent yolk plugs.

(c) 3 hours in LiCl, plus 79 hours in water. Microcephalic
to anencephalic embryos with ‘fused’ suckers and olfactory pits. There
are probably some cyclopic embryos among these. Figure 47 illus-
trates one of the more extreme types obtained under these conditions.
There is a single olfactory pit and the ventral suckers are reduced to a
single, small, cone-like protuberance.

(d) 3 hours in LiCl, plus 103 hours in water. Development
has continued and the inhibiting effects of the treatment are more
marked. All of the embryos are now much distended ventrally by an
accumulation of fluid in the coelome or possibly in the pericardial sac.
If punctured ventrally with a needle some of the fluid comes away and
the swelling recedes slightly for an hour or so when, shortly, the dis-
tention becomes as great asever. Figures 48, 49 illustrate two of these
embryos. In a number of cases (probably one-fourth), while the
formation of an embryo has proceeded to some extent, one does not
readily distinguish, macroscopically at least, between anterior and

MODIFICATION OF DEVELOPMENT IN THE FROG 489

posterior ends. Except for this apparent lack of anteroposterior dif-
ferentiation, they remind one just a little of a teleost egg. One of these
peculiar embryos was isolated (fig. 50) because it had, at the time, the
appearance of being bipolar, i.e., possessing either two heads or two
tails, one at either end of the ‘embryonic area.’ Several days later,
however, it developed into an almost anencephalic embryo with a more
or less characteristic tail bud (fig. 51).

Us,
ee
50

48
4 f oe —a
epee fs SE * = >
ihe Cl Fe fe : Senn =
a eS STEEN
te tex HY if af Tee a
id
Ped Sf 5 3 E <I
Aarts lees “nce Pe
oe fin =a here 2a)
iB Po yo Sd eee ae 4
se he ae peat neice :
Me MeLioes ess aa)
aOR i esecae Sarae
Sie sy < PADS 2
aN Piso Sear a
x peterestee =
ee ates) ‘4 oi
Seep
ae tars ee 5]

Figs. 47 to 51 Differential inhibition. m/5 LiCl. Eggs late cleavage stage
when placed in the solution. Treatments: 47, 3 hours in LiCl + 79 hours in
water. Lateral view (anterior end at top) of an extreme type showing little
regional differentiation. 48, 49, 3 hours in LiCl + 103 hours in water. Shows
some recovery. 90, 51, see text, page 489. Exp. IV 58. o.p., olfactory pit; v.s.,
ventral sucker.

oO

kK—7m—

Lot D. (a) 5 hours in LiCl, plus 4 hours in water. The eggs are
in much the same condition as those in lot B (a).

(b) 5 hours in LiCl, plus 47 hours in water. Large yolk
plug stages or equatorial gastrulae and in many cases showing a second-
ary invagination above the equatorial blastopore. An egg of this sort
was figured (fig. 18) in the previous report.

490 A. W. BELLAMY

[ita

TASES
Se

ORD
=

es

ie
Les

60

59
Figs. 52 to 60 Differential inhibition. 52, 53, outline views of same embryo
shown in figures 1, 2, 16 1/2 hours later. 54, 55, dorsal views of two embryos and,
56 to 60, lateral views of five embryos after treatment in m/5 LiCl. Eggs late
cleavage when placed in the solution. Treatments: 54, 55, 53 hours in LiCl.
56 to 60, 53 1/2 hours in LiCl + 10 days in water. Exp. IV 59. 6b, open brain
region; v.s., ventral sucker; y.p., yolk plug.

MODIFICATION OF DEVELOPMENT IN THE FROG 49]

(c) 5 hours in LiCl, plus 77 hours in water. Generally no
advance—dead or dying as equatorial gastrulae.

-Lot E. (a) 18 hours in LiCl, plus 34 1/2 hours in water. No ad-
vance over Lot A (a).

Experiment IV 59. Eggs from the same batch and of the same age
as those used in experiment IV 58 were placed in m/10 LiCl. Eggs
removed to water at intervals of 1 1/2, 3, 5, 24, and 53 1/2 hours.

Lot A. (a) 18 hours in LiCl. Late, inhibited gastrulae to early
yolk plug stages.

(b) 53 hours in LiCl. Early neural fold stages, most of
which have a long narrow yolk plug—a consequence of the relatively
greater inhibition of the dorsal lip region. Figure 1, dorsal and 2,
lateral views of a single embryo, shows an oblong yolk plug extending
over approximately 90°. Figures 52, 53 show in outline two views of
the same embryo 16 1/2 hours later. The embryo is microcephalic,
ventral suckers approximated but not entirely ‘fused’ and with the
neural groove open through most of its length. Figures 54, 55 show
two similar embryos from the same lot.

(c) 93 hours in LiCl. All degrees of approximation to com-
plete ‘fusion’ of ventral suckers and olfactory pits. All of the embryos
have permanent yolk plugs. Many are nearly or quite anencephalic.
Others show the anterior part of the neural groove open and undergoing
disintegration (figs. 3, 4, 5, 6).

(d) 107 hours n LiCl. No further advance. Almost all

dead.

Lots B, C, D. Eggs exposed 1 1/2, 3 and 5 hours to this solution
and then returned to water, hatched s'multaneously or slightly behind
the control, showing no marked effects of the treatment.

Lot E. (a) 24 hours in LiCl, plus 28 1/2 hours in water. Some
differential inhibition as shown by the relatively greater retardation of
the dorsal lip region. Neural folds just closing. A small yolk plug
is still present.

(b) 24 hours in LiCl, plus 57 1/2 hours in water. Hatching
slightly behind the control. Almost complete recovery. Tails seem
relatively a little larger than in control.

Lot F. (a) 53 1/2 hours in LiCl, plus 28 hours in water. Marked
differential inhibition. The embryos show all degrees of approximation
to complete ‘fusion’ of ventral suckers and olfactory pits.

(b) 53 1/2 hours in LiCl, plus 114 hours in water. Micro-
cephalic embryos with ventral suckers, olfactory pits, and optic vesicle
approximated or ‘fused.’ All of the embryos show varying degrees of
differential recovery as indicated by the dorsal convexity and dispro-
portionately large tails. Figures 56 to 60 show five of the embryos
after 10 days in water.

Figs.61 to 70 Differential inbibition. 61 to 63, lateral views of three embryos
after 14 hours in m/5 LiCl and 5 days in water. Eggs in early gastrula stages
when placed in the solution. The greater inhibition here appears to be a
consequence of the higher susceptibility of the eggs at the time of gastrulation.
Exp. 121.1 E. 64, 65, dorsal views of two embryos after 24 hours in m/5000
KNC and 5 days in water. Eggs in two-cell stage when placed in the
solution. Microcephalic with shallow neural groove and persistent yolk
plug. Exp. KNC-C.8a. 66, a spina bifida and 67, 68, two other embryos
after 4 days in m/10,000 KNC and 8 days in water. Eggs in two-cell stage
when placed in the solution. Same experiment as embryo shown in figure
23. 68 is shown with the anterior end twisted somewhat to one side, to show the
almost completely united ventral suckers. 69, 70, two embryos after 4 days in
KCN + 8 days in water. Less than 1 per cent of the embryos in this experiment
showed this extreme inhibition. Exp. KNC-C.10. m, mouth; 0, olfactory pit;
t, tail; v.s., ventral sucker; y.p., yolk plug.

492

MODIFICATION OF DEVELOPMENT IN THE FROG 493

Potassium cyanide

Eggs in a two-cell stage at the beginning of the experiment (experi-
ment KNC-C.9°). The eggs were divided into two lots and treated
as follows in m/10,000 KNC:

Lot A. (a) 48 hours in KNC. Equatorial gastrulation beginning
in a few eggs. Control eggs show advanced yolk plug stages.

(b) 4 days in KNC. More than 90 per cent of the eggs
show a circular blastopore following the equatorial region (equatorial
gastrulae). The controls are now elongating embryos with heads
formed.

(c) 5 days in KNC. Little if any change. The eggs con-
tinued in this solution without further advance in development until
the eighth day, when they began dying in considerable numbers.

Lot B. (a) 4 days in KNC, plus 2 days in water. Mostly neural
fold stages with relatively small head region and showing a persistent .
yolk plug. Figures 10, 11 illustrate one of the less extreme embryos
of this type. Most of the embryos show a much larger yolk plug and
situated more dorsally and also showing greater inhibition of the head
region. Comparison with figure 55 reveals a striking resemblance in
the type of abnormality produced with the aid of two widely different
chemical agents—potassium cyanide and lithium chloride.

(b) 4 days in KNC, plus 5 days in water. Mostly micro-
cephalic forms showing spina bifida and with persistent yolk plugs.
All of the embryos show various degrees of approximation to complete
‘fusion’ of ventral suckers and olfactory pits. Figure 23 illustrates
one of these embryos. It will be noticed that the yolk plug is situated
dorsally and that the two tails curve upward with the distal ends di-
‘rected toward the anterior end.

(c) 2 days in KNC, plus 8 days in water. Spina bifida of
various sorts. Figure 66 illustrates one of these, showing in addition
a microcephalic condition and a single ventral sucker. This is repre-
sentative of the lot at this age and resembles somewhat the embryo
illustrated in figure 23. Figures 67, 68 are later stages of the few less
extremely modified embryos drawn to the same scale. Figure 68 is a
ventrolateral view of one showing the ventral suckers almost completely
‘fused’ and a single olfactory pit. Probably 5 per cent of the embryos
in this lot were similar to 67, 68, the remaining embryos approximating
the type shown in figure 66.

This experiment is typical of those furnishing conditions mentioned
at the beginning of the description of the experiment. The range of
concentrations of KNC that will produce considerable deviations from
the normal and still permit development to proceed beyond gastrula
stages is rather limited. In m/1000 development stops in mid-cleavage
stages with the pigmented and unpigmented cells approximating the
same size. Likewise in m/5000 development stops during cleavage

6 Citations of experiments in italics refer to those done by Professor Child in
1916.

494 A. W. BELLAMY

stages—usually late cleavage stages. The eggs have much the same
appearance as those treated in m/1000 KNC. The eggs will continue
development, however, if they are removed to water or to lower con-
centrations of cyanide. For instance, in experiment KNC-C.6,
where eggs undergoing the first cleavage were exposed to the follow-
ing concentrations of KNC: m/5000 24 hours, m/10,000 24 hours,
m/20,000 24 hours, m/50,000 12 hours, reached late yolk plug stages.
The yolk plugs were large and protruded considerably. In many cases
the yolk plugs were: elongated in the median plane, i.e., elliptical in
outline. Even with the preliminary treatment of 24 hours in m/1000
KNC, then 24 hours in m/20,000, 12 hours in m/50,000, and 24 hours
in m/100,000 (experiment KNC H6—eggs from same lot as above),
equatorial gastrulae are formed somewhat similar to those produced
in m/500,000 HgCl,—24 hours in the solution and 24 hours in water,’
and to those produced in LiCl—48 hours’ exposure to m/10. A few
eggs from this lot were similar to the ‘secondary invagination’ forms
obtained frequently in m/10 LiCl after 48 hours’ exposure.’ In
m/20,000 KNC development is retarded and the inhibition is differ-
ential, but very few extreme modifications are seen.

Potassium cyanide

Experiment ANC-C. 10. Eggs from the same female and in two-
cell stage were divided into two lots and treated as follows in m/20,000
KNC:

Lot A. (a) 48 hours in KNC. Mostly advanced yolk plug stages,
much like control. There is, however, a slight retardation of the dor-
sal lip region.

(b) 4 days in KNC. The embryos range all the way from”
late blastula and early gastrula stages to closed blastopore and early
neural fold stages—mostly the latter. A few embryos of the spina
bifida type. Control: elongated embryos with distinct heads.

(c) 6 days in KNC. Blastopore has closed in most of the
embryos. Body still round or only beginning to elongate. A few
spina bifida present.

(d) 8 days in KNC. Nearly normal in form, though develop-
ing slower than control. Many dying after hatching. Ten to 20 per
cent of the embryos spina bifida.

(e) 9 days in KNC. Little or no further advance. Sixty
to 70 per cent dead or dying. Heads range from about normal to
distinctly small or retarded.

Lot B. (a) 4 days in KNC, plus 2 days in water. Nearly all of
the embryos elongating; head becoming distinct. All less advanced
than control. Many of the embryos show differential recovery dis-
tinctly as shown by the relatively advanced condition of the head and
in some case of the tail buds.

7 See figure 14, previous report.
8 See previous report, figure 18.

MODIFICATION OF DEVELOPMENT IN THE FROG 495

(b) 4 days in KNC, plus 4 days in water. Seventy per cent
hatched, others dead in membranes without elongating. The hatched
animals seem to have rather large heads.

(c) 4 daysin KNC, plus7 daysin water. Actively swimming
embryos assuming the regular tadpole form. Smaller than control,
but with relatively larger heads—showing a differential recovery.

(d) 4 days in KNC, plus 8 days in water. Nearly all nor-
mal tadpoles except that the heads are relatively larger. A few spina
bifida and microcephalic forms remain alive. Figures 69, 70 illustrate
two of the very few—about 1 per cent—extremely modified embryos.
They are microcephalic and bent dorsally. In one, shown in 80, the
ventral suckers are completely ‘fused,’ but only partly fused in the
other, 81.

If the eggs are placed in m/10,000 KNC in late gastrula stages, de-
velopment proceeds slowly for several days and ceases with the embryos
dying in early neural fold stages.

In m/50,000 and m/100,000 KNC there is usually some acceleration
of development and many of the embryos appear somewhat megace-
phalic. The acceleration is not as great nor is the differential nature
of the acceleration as marked as in certain concentrations of HCl (p.497).
Figure 29 shows a camera-lucida outline of one of the more extreme
megacephalic types.

Experiments with formaldehyde

Experiment IV 62. Eggs in four to eight-cell stages were treated
as follows in 0.0075 per cent formaldehyde.

12 hours. Early cleavage stages with pigmented and unpigmented
cells approximating the same size.

24 hours. Early inhibited gastrulae.

48 hours. A few early neural fold stages and these with very shallow
neural grooves and with persistent yolk plugs. Figures 12, 13 illustrate
two of these.

72 hours. 95 per cent spina bifida. Ventral suckers approximated
(fig. 14). There appears to be some acclimation especially in the head
region.

133 hours. Not much change. Many dying.

A number of concentrations of formaldehyde were tried ranging from
0.01 to 0.001 per cent, but the concentration used in experiment IV 62
was the only one that gave any considerable number of markedly modi-
fied embryos in the later stages of development. ‘The percentage strength
was calculated on the basis of 40 per cent commercial formalin. For
example, the 1 per cent (approximate) stock solution from which the
other strengths were made up was made by adding 1 ce. of the commer-
cial product to 39 cc. of water.

496 A. W. BELLAMY

Experiments with HCl

In all of the experiments involving HCl and NaOH the solutions were
made up from freshly prepared (in the case of HCL) n/10. These stock
solutions were diluted with an amount of well-water that would give,
theoretically, the strength of acid or alkali stated for each experiment.
And while, in the case of HCl the concentration of acid was certainly lower
than that stated due to the presence of carbonates and salts in the well-
water, the solutions were always prepared in the same way, so that,
regardless of the actual concentration or of the hydrogen ion-concen-
tration, the figures representing the strength of acid are sufficient for
purposes of comparison. It will be understood, then, that the con-
centration given, e.g., n/5000, is relative, indicating merely the amount
of n/10 stock solution added to a given quantity of well-water necessary
to furnish a theoretical n/5000 solution. We hope later to report a.
series of experiments similar to these in which the actual hydrogen-ion
concentration is known. In all experiments liter Erlenmeyer flasks
were used. They were filled with the solution and stoppered. Con-
trols were run parallel in stoppered flasks of well-water. Solutions
were changed daily unless otherwise noted.

Figures 15 to 21 illustrate seven embryos from experiment 130.3.
The eggs when in early gastrula stages were placed in n/750 HCl and
after twelve hours were removed to water. The figures show their
condition eighty-one hours later. They show just about the same
abnormalities and the same range of variation in type as is seen after
treatment with a great variety of other agents and conditions. EKm-
bryos of much this same type were obtained in three other series of
HCl experiments, furnishing approximately the conditions noted above.

One curious consequence of the HCl treatment in low concentrations
was a very marked acceleration of development. Without attempting
to decide at present whether the acceleration is due directly or indirectly
to the HCl as such, the results are perfectly clear cut and definite.
Both Professor Child and myself have obtained these acceleration
forms independently and repeatedly. Furthermore, there is a ten-
dency for the embryos to become megacephalic under such conditions
and often the tails, ventral suckers, and gills are unusually prominent.
In other words, the acceleration is more or less differential. The em-
bryo apparently does not simply develop more rapidly as a whole,
but certain regions are more accelerated than others, viz., the same
regions that are most inhibited under conditions that markedly retard
developmental processes.

Experiment HCI-C. 1. 1/5000 HCl. Eggs two to four-cell stage
at beginning of the experiment.

30 hours in HCl. Early gastrula. In advance of control.

60 hours in HCl. Neural fold stages. Control, small yolk plug
stages (figs. 30, 31).

72 hours in HCl. Blastopore closed and embryos beginning to
elongate. None of the control embryos elongating yet.

MODIFICATION OF DEVELOPMENT IN THE FROG 497

96 hours in HCl. Embryos in advance of control. Heads seem
relatively larger than in normal embryos (figs. 32, 33, 34).

Experiment HCI-C. 5. n/5000 HCl. Eggs two to four-cell stage at
the beginning of the experiment.

48 hours in HCl. Distinctly accelerated. Blastopore closed and
medullary folds distinct. Control: advanced yolk plug stages.

96 hours in HCl. Many hatched. Heads seem relatively large.
~ None of the control embryos hatched as yet.

6 daysin HCl. Much accelerated. Tails long and broad. Figures
35, 36, 87 and 38. The heads of these embryos appear relatively longer
and broader than controls and the gills are further developed and wide-
spread. Actively swimming.

Experiment HCI-C. 6. n/20,000 HCl. Eggs two to four-cell stage
at the beginning of the experiment. Control same as for the experi-
ments just described.

48 hours in HCl. Development accelerated. Blastopore closed.
Medullary folds distinct and beginning to close. Dorsal region elongat-
ing.

96 hours in HCl. Embryos are more accelerated than those in
n/5000. Nearly all hatched.

9 days in HCl. Heads relatively large and gills unusually promi-
nent. The embryos remind somewhat of Amblystoma tadpoles.

The experiments with NaOH were carried out in the same way as
those with HCl, and in most cases were ran parallel with eggs from
the same female and one control for the two series. Save for the slightly
different concentrations used, there was no particular difference in
the experiments with the two agents either as regards method or re-
sults. A single example will suffice here. Eggs in early gastrula
stages exposed from 1 1/2 to 12 hours to n/300 NaOH and returned to
water show after several days microcephalic embryos with the bilateral
sense organs and ventral suckers approximated or ‘fused’ and numerous
spina bifida forms are present. Figures 24 to 26 represent three em-
bryos from experiment 125.2 E. The eggs—early gastrula at the
start of the experiment—were exposed 3 hours to n/300 NaOH and
removed to water. The camera-lucida sketches were made six days
later.

If eggs are taken when the susceptibility is low—early cleavage or
unsegmented—and somewhat lower concentrations of NaOH used,
n/400, n/500—n/1000, development is accelerated in much the same
way asin the HCl experiments described above. Figures 27, 28 repre-
sent two sister embryos of the same age. Figure 27 is the control and
28 an embryo exposed 8 days to n/500 NaOH. In this case the solu-
tion was not changed after the third day. The megacephalic condi-
tion—differential acceleration—is apparent at once.

As regard acclimation of the egg or embryo in the various solutions
my data are incomplete, but a number of the experiments furnished
conditions where recovery might be expected to take place. Potassium
permanganate seems to be a favorable agent for the purpose. While

498 A. W. BELLAMY

quite toxic in concentrations of m/5000 or higher, the toxicity appears
gradually to grow less, due to the formation of the insoluble oxide.

Eggs in an eight-cell stage were placed in m/5000 potassium per-
manganate, and after 48 hours’ exposure a series of stages was present,

varying from nearly normal yolk plug stages to equatorial gastrula
stages. The controls at this time were in neural fold stages. After
72 hours most of the embryos were of the type designated as differen-
tial recovery forms. The heads are relatively very large and the ven-
tral suckers and gill plates are prominent. At this time there still
remained alive a few equatorial gastrulae and several spina bifida forms,
microcephalic and with the persistent yolk plug situated dorsally.
Variations of this sort can hardly be avoided in material such as the
frog egg for reasons already set forth. Moreover, the thickness of the
gelatinous membranes varies somewhat, especially after the large egg
masses have been cut apart into smaller masses containing ten or a
dozen eggs, as was done in all of the experiments. In this particular
experiment approximately 75 or 80 per cent of the embryos were of the
differential recovery type, so that on the whole the results are perfectly
clear-cut and definite.

Figures 41, 42, which are described on page 486, represent what
appear to be variations in recovery for which the factors mentioned
above are, I believe, in part responsible. In addition, the conditions
under which these embryos were produced were severe enough that
recovery rarely occurs at all. Consequently, some variation is to be
expected. But whatever the extent of the variation may be, the signifi-
cant point is that for the most part, recovery following a previous
inhibition is differential. Figure 44 illustrates differential recovery
following a previous treatment with lithium chloride.

It is not always possible to distinguish between acclimation and
recovery, especially under conditions like the permanganate experi-
ment cited above. In this case the strength of the solution is gradually
changing, due to the reduction of the permanganate (the solution was
not changed during the course of the experiment). But little or no
acclimation occurs under the conditions which produced some of the
embryos. Hence, in this case the results are in all probability a conse-
quence of the earlier inhibition and later recovery. Many of the em-
bryos were convex dorsally and the tail buds were relatively large and
Maen Similar embryos appeared after the following treatment :

2 days in water (blastopore closed and medullary folds appearing at
this time), plus 2 days in m/10,000 KNG, plus 2 days in water. The
differential was even more marked in these embryos (experiment KNC-
CxaiB):

Recovery of the embryo, then, is differential in the same sense that
inhibition is differential. Those regions of the embryo that are nor-
mally most active, recover soonest when the developing animal is
removed from the inhibiting conditions.

MODIFICATION OF DEVELOPMENT IN THE FROG A499

THE INTERPRETATION OF ABNORMAL DEVELOPMENT

Quite apart from any interpretation that may be given to the
facts, the egg and embryo of the frog exhibit a differential sus-
ceptibility to various factors of the environment. ‘That is, those
regions of the egg or embryo that are functionally most active
are most susceptible to agents or conditions that at all seriously
disturb developmental processes. This is only another way
of saying that these differences in susceptibility bear a direct
relation to the principal axes of symmetry of the egg and embryo.
The different methods of demonstrating differential susceptibility
have already been discussed.

Given what may be called gradients in susceptibility which
is demonstrated, it follows as a necessary consequence that any
considerable disturbance of development, experimentally or
otherwise induced, save certain mechanical disturbances of course,
will appear as a differential. Hence, in the absence of specific
effects of particular agents or conditions on particular regions of
the egg or embryo, inhibition, acceleration, acclimation, or re-
covery in development must be differential. As a matter of
fact, inhibition that is at all marked is differential, regardless of
how it is induced. The same is true to a considerable extent of
accelerated development and in those cases where the egg or em-
bryo acclimate to or recover from some disturbing condition.

If additional evidence is needed to convince one of the dif-
ferential nature of inhibited development in the frog, it can be
had by examining the data of previous workers in this field—
Gurwitsch, 96; Hertwig, ’92, 95; Bataillon, 01; Morgan, ’03;
Jenkinson, ’06; these are a few of the representative papers.
The great majority of modifications in the development of the
frog obtained by these other workers in the field are so obviously
differential inhibition forms that a detailed discussion of them
seems quite unnecessary.

It is significant that practically all, certainly the most common
types of abnormalities and especially those experimentally in-
duced not only in the frog, but also in organisms generally, fall
naturally into the categories mentioned earlier in the paper, viz.,
differential inhibition forms, or more rarely, since the conditions

500 A. W. BELLAMY

for their appearance are less often realized, abnormalities showing
differential acclimation or recovery, or differential acceleration.

Often the embryo shows a combination of inhibition and aceli-
mation or recovery or both. The failure to distinguish these
different types when seen in the same embryo may lead to some
confusion in attempting to interpret abnormal development. I
do not, of course, intend to imply that all abnormalities are
consequences of conditions which, operating relatively early in
development, produce differential inhibitions, accelerations,
acclimations, or recoveries. Mechanical disturbances and the
like and probably other disturbances coming near the end of de-
velopment belong to quite a different category. With the great
majority of teratological forms falling readily into the classes
mentioned above or combinations of them which in turn must be
consequences of differential susceptibility, the task of interpret-
ing teratological development resolves itself largely into a matter
of accounting satisfactorily for differential susceptibility.

The reasons for regarding differential susceptibility as one
expression of underlying graded differences in the dynamic re-
lations and activities of protoplasm—physiological gradients—
have been presented elsewhere (Child, ’20; Bellamy, 719).

Given the organismic pattern as a gradient or gradients in
fundamental functional and structural relationships in a specific
protoplasm, and there is abundant experimental evidence to sup-
port this idea, teratological development becomes understandable
as a disturbance of this underlying order. Since this order is a
graded one paralleling the axes of symmetry of the organism, any
considerable disturbance of it must also entail a parallel and
differential modification in the development of the embryo.

SUMMARY

1. The egg and embryo of the frog exhibit a differential sus-
ceptibility to a great variety of conditions that disturb develop-
mental processes.

2. According to the experimental treatment and the physio-
logical condition of the organism at the time of exposure, the
induced modifications fall naturally into classes of differential

MODIFICATION OF DEVELOPMENT IN THE FROG 501

inhibitions, differential accelerations, differential acclimations,
or differential recoveries.

3. These differential modifications, are obvious and necessary
consequences of the differential susceptibility of the organism
to the agents used in experimental teratology.

4. The modifications produced are perfectly characteristic, not
of a particular agent or condition, but of a particular concentra-
tion or ‘intensity’ of action of that agent. In other words, the
developmental response is primarily quantitative and non-
specific.

5. Differences in susceptibility parallel the axes of symmetry.
Those regions which usually differentiate earliest and grow most
rapidly are most distorted, the degree and direction of the dis-
tortion depending largely upon the severity of the treatment and
physiological condition of the organism. There are, in short,
gradients in susceptibility paralleling the axes of symmetry, both
primary and secondary.

6. The existence of susceptibility gradients—whose existence
is demonstrated in a great variety of organisms—must mean an
underlying gradient or gradients in the structural and functional
aspects of protoplasm generally.

7. It isin terms of disturbances in this underlying graded order
in the dynamic relations and activities of protoplasm—physiolog-
ical gradients—that we believe teratological development is most
rationally accounted for.

LITERATURE CITED

BartarLton, E. 1901 Etudes experimentales sur l’evolution des Amphibiens.
Arch. Entw.-Mech., Bd. 12, 8. 610-655.

Betiamy, A.W. 1919 Differential susceptibility as a basis for modification and
control of early development in the frog. Biol. Bull., vol. 37, pp.
312-361.

Cuitp, C. M. 1916 Experimental control and modification of development in
the sea-urchin in relation to the axial gradient. Jour. Morph., vol. 28,
pp. 65-133.
1920 Some considerations concerning the nature and origin of physio-
logical gradients. Biol. Bull., vol. 39, pp. 147-187.
1921 The origin and development of the nervous system. Chaps. 1-5.
The University of Chicago Press.

502 A. W. BELLAMY

Gurwitscnu, A. 1896 Ueber die formative Wirkung des verinderten chemischen
Mediums auf die embryonale Entwickelung. Arch. Entw.-Mech.,
Bd. 3, S. 219-260.

Herrwic, O. 1892 Urmund und Spina bifida. Arch. f. mikrosk. Anat., Bd.
39, 8. 353-503.
1895 Beitriige zur experimentellen Morphologie und Entwicklungs-
geschichte. 1. Die Entwicklung des Froscheies unter dem Einfluss
schwiichere und _ stiirkerer IKochsalzlésungen. Arch. f. mikrosk.
Anat., Bd. 44, 8. 285-344.

Jenkinson, J. W. 1906 On the effects of certain solutions upon the develop-
ment of the frog’s egg. Arch. Entw.-Mech., Bd. 21, 8.367460.

Morean, T.H. 1903 The relation between normal and abnormal development
of the embryo of the frog as determined by the effect of lithium chloride
in solution. Arch. Entw.-Mech., Bd. 16, 8. 691-712.

Spemann, H. 1918 Uber die Determination der ersten Organanlagen des
Amphibien-embryo. I-VI. Arch. Entw.-Mech., Bd. 43, S. 448-555.

Srockarp, C. R. 1921 Developmental rate and structural expression: An
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and the interaction among embryonic organs during their origin
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the sea-urchin in relation to the axial gradient. Jour. Morph., vol. 28,
pp. 65-133.

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Resumen por el autor, A. L. Salazar.

Sobre una forma particular de atresia de los foliculos de De Graaf
(coneja) revelada por el método tano-argéntico.

El autor describe un tipo especial de atresia de los foliculos
graafianos en el ovario de la coneja, revelada por su método.
Este tipo se observa en los pequefios folfculos del ovario de tipo
ovigénico, caracterizindose por la formacién de un largo cordén
tanofflico en los intersticios de las células de la granulosa. El
autor examina la conexién de este tipo de atresia con la atresia
hidrépica de los cordones ovigénicos y sus restos (nédulos epi-
teliales, folfeulos aovdricos de Regaud), recientemente descritos
por él, y discute la significacién de los casos descritos.

Translation by José F. Nonidez
Cornell Medical College, New York

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MAY 1

SUR UNE FORME PARTICULIERE D’ATRESIE DES
FOLLICULES DE DE GRAAF (LAPINE), REVELEE PAR
LA METHODE TANNOFERRIQUE

A. L. SALAZAR
Institut d’Histologie et d’Embryologie de Vv Université de Porto, Faculté de Médecine
(Portugal)

CINQ FIGURES

Dans les ovaires adultes de la Lapine, appartenant au type
ovigéne, on trouve un certain nombre de formes atypiques d’atré-
sie folliculaire. Parmi ces formes, les follicules 4 cordon tanno-
phile présentent un intérét spécial 4 cause de leurs rapports avec
Vatrésie hydropique, que nous avons signalée récemment?! dans
Vovaire de la Lapine. Nous allons décrire ici quelques types de
follicules 4 cordon tannophile; ensuite nous étudierons leurs rap-
ports avec ’hydropisie atrésique des cordons ovigénes et de leurs
reliquats. Cette étude est basée sur des coupes traitées par notre
procédé au tannin-fer, suivant la technique que nous avons
indiquée ailleurs.?

A. LES FOLLICULES A CORDON TANNOPHILE

Le type d’atrésie en question est caractérisé par l’existence
dans les interstices entre les cellules de la granulosa ou dans la
place totale que celle-ci occupait, d’un long cordon plus ou moins
épais, coloré en noir intense par la réaction en question. Ce
cordon présente comme caractéristiques générales l’intense tanno-
philie, la longueur et des sinuosités innombrables; il remplit
parfois tout le follicule, dont la granulosa est invisible ou réduite
a quelques cellules. Ces follicules 4 cordon tannophile peuvent
Sse présenter avec quelques variations: nous représentons dans
les figures 1, 2 et 3, trois spécimens. Dans les coupes traitées par
la méthode tanno-ferrique ces follicules, comme d’ailleurs tous
les ovisacs dans la période agonique de l’atrésie, se détachent

503

THE AMERICAN JOURNAL OF ANATOMY, VOL, 30, NO. 4

504 Ae By. SATA

nettement en noir intense sur le fond blanc ou grisatre de la pré-
paration, ot seules les formations tannophiles (pellucide, liquor
folliculi, membranes de Slavjanski et des cordons, cordons ovi-
génes et reliquats en atrésie hydropique, etc.) se dessinent en
noir.

Dans la figure 1 on voit un follicule encore peu développé. II
est limité par une membrane de Slavjanski intacte, que le tannin-
fer dessine nettement, comme d’habitude, sous la forme d’un
mince trait noir. Cette conservation de la membrane de Slavy-
janski est particuliérement remarquable étant donnée la tendance
de cette membrane a s’hypertrophier dans l’atrésie typique et
méme dans l’atrésie des cordons ovigénes et des reliquats.? Au
centre du follicule, teinté en gris, on trouve l’oocyte dégénéré.
Dans la coupe représentée dans la figure on ne voit pas la pellu-
cide; elle était visible dans les coupes suivantes, gonflée trés
tannophile, comme c’est, du reste, le cas habituel dans les périodes
agoniques de l’atrésie.

De la granulosa on ne voit qu’une rangée de cellules super-
ficielles, dont les noyaux sont 4 peine visibles, car le tannin-fer ne
colore pas la chromatine. La portion restante de la granulosa
est occupée par un cordon tannophile assez épais, trés long,
intensivement coloré en noir. Ce cordon présente partout sensi-
blement la méme épaisseur, 4 l’exception d’un point ot il se
gonfle en une grosse boule noire, qu’on voit dans la figure; cette
boule semble une hypertrophie locale du cordon. Celui-ci pré-
sente partout des flexuosités et des circonvolutions si nombreuses
et si serrées qu’il est impossible de le suivre et de savoir s’il s’agit
d’un cordon unique ou de plusieurs cordons enchevétrés. Il
s’agit probablement d’un cordon unique, car on ne voit nulle part
des solutions de continuité. Nous avons taché de représenter
par les différentes graduations de ton l’aspect que présente dans
la coupe, assez épaisse, ce cordon capricieusement pelotonné.
L’épaisseur de la coupe permetait la représentation du cordon
en plusieurs plans. La rangée de cellules de la granulosa, qu’on
voit dans la périphérie, sous la membrane de Slavjanski, semble
intacte; mais le tannin-fer, ne colorant ni le noyau ni le cyto-
plasme, il est impossible de l’affirmer avec certitude. Cependant,

FOLLICULES DE GRAAF A CORDON TANNOPHILE 505

la forme du noyau,a peine visable par réfringence, est réguliére, et
il n’y existe pas des phénoménes de plasmolyse ni de ecaryolise.
Le liquor interstitiel? est invisible; on apercoit débris dans le
voisinage de la pellucide dégénérée. Cette absence du liquor
interstitiel peut étre réelle ou apparente; car, dans ces cas aty-
piques, sa tannophile est parfois si faible qu’elest souvent difficile-
ment visible. Les cellules de la granulosa qu’on voit dans la
figure disparaissent dans les coupes suivantes; dans ces coupes le
follicule en question est entiérement occupé, outre l’oocyte et la
pellucide dégénérée, par le cordon tannophile. Nous avons
examiné soigneusement les interstices entre les flexuosités du cor-
don, pour chercher les cellules de la granulosa ou leurs débris;
nous n’avons réussi 4 voir rien de net, car l’enchevétrement du
cordon est si compliqué, qu’on n’apercoit entre les flexuosités que
l’ombre dense des autres régions du cordon.

La figure 2 représente un autre follicule atrésié avec cordon
tannophile. Il s’agit d’un follicule un peu plus développé que le
précedent. L’oocyte, excentrique, se trouve réduit 4 des frag-
ments; la pellucide, gonflée, retractée, présente une bande interne
trés tannophile, colorée en noir intense, et une région externe
colorée en gris de plus en plus pale au fur et 4 mesure qu’on
s’approche de la périphérie, oti elle se termine par des estompés
trés larges. Elle présente une striation concentrique un peu
vague. C’est, en somme, un des aspects habituels de cet élément
dans les follicules atrésiés: elle se trouve en pleine deliquescence
atrésique. Le follicule est limité, comme le précédent, par une
membrane de Slavjanski dessinée sous la forme d’un mince trait
noir. Ce trait noir, trés net et indépendant en certains points,
s’estompe en d’autres, oti il se confond tantét avec le dessin du
liquor interstitiel, tantét avec le conjonctif environant, tanté6t
enfin avec l’un et l’autre 4 la fois.

La granulosa est visible en partie. On voit des-plages occupées
par des cellules, dont on apercoit vaguement les noyaux et nette-
ment les contours dessinés par le liquor interstitiel. La plus
grande partie de la granulosa a été substituée par un trés long
cordon tannophile, qui ondule partout. Ce cordon présente une
portion qui dessine a la périphérie du follicule des spires de cou-

506 A. L. SALAZAR

leuvre; la portion restante, la plus longue, présente des sinuosités
si sérrées, qu’elles se touchent par leur dos. L’aspect n’est si en-
chevétré que dans le follicule précédent, car les sinuosités suivent
presque toujours la ligne du cordon et cette ligne présente dans
son ensemble des ondulations trés larges. Ici, comme dans le
cas présédent, le cordon semble continu, car on n’y voit pas de
solutions de continuité. Dans les coupes on voyait plusieurs
follicules atrésiés présentant un cordon moins long, en général
fragmenté, passant par des transitions insensibles a la substance
tannophile qui s’accumule en lacs dans les interstices entre les
cellules de la granulosa atrésiée. On pourrait échelonner toute
une serie d’exemplaires depuis ceux qui présentent un cordon
trés long, comme celui de la figure 1, Jusqu’A ceux qui n’en pos-
sédent que quelques fragments. Ces follicules 4 cordon plus
réduit et fragmentaire occupent le centre de f. c. j. en formation,
au contraire de ceux que nous venons de décrire. En effet, ceux-
ci ne sont pas suivis de la formation de corps jaunes atrétiques ce
qui est, du reste, le cas habituel dans l’atrésie des follicules trés
petits. Les follicules atrésiés avec cordon petit, irrégulier, frag-
menté, existent d’ailleurs dans les ovaires du type interstitiel,
atrésique-interstitiel, etc., au contraire des formes précédentes,
que nous n’avons rencontrées jusqu’ici que dans l’ovaire du type
ovigéne. Nous n’insisterons pas pour le moment sur ces formes
attenuées, c’est-d-dire 4 petit cordon fragmenté, qui établissent
une transition entre les formes atypiques (ou mieux rares) et
Vaspect du follicule agonique coloré par le tannin-fer; nous y
reviendrons avec plus de détails dans un travail sur les moments
agoniques de l’atrésie du follicule et la formation du corps jaune
atrétique. Nous voulons simplement faire remarquer qu’entre
les formes les plus atypiques représentées dans les figures 1, 2 et 3
et les cas typiques habituels il existe une série graduelle de formes
de transition.

Le cas représenté dans la figure 3 appartient encore 4 la caté-
gorie des précédents, mais il s’en distingue par quelques caractéres
particuliers. Le follicule présente la forme d’une grosse poire.
La portion grosse représente le follicule proprement dit; le pédi-
cule est un fragment du cordon ovigéne resté en connexion avec

A

FOLLICULES DE GRAAF A CORDON TANNOPHILE 507

Yovisac. Ce fait est trés fréquent, surtout dans les ovaires du
type ovigéne, mais il présente dans ce cas un aspect curieux.
D’abord, l’appendice est constitué par une seule rangée de cellu-
les; puis, il est si long, que nous n’avons pu le représenter qu’en
partie dans le dessin; nous n’avons pu d’ailleurs le suivre jusqu’a
son terminus, tant il était long. Un examen plus détaillé montre
lesuivant. la portion grosse, c’est-a-dire le follicule proprement
dit, présente deux pellucides en déliquescence atrésique. Le
centre des deux pellucides est occupé par des débris de l’oocyte
respectif. Les deux pellucides présentent une portion interne
homogéne, trés tannophile, et une région externe, plus étendue,
plus pale; cette région externe est, du reste, commun aux pel-
lucides, ce qui est dd A la liquéfaction. D’ailleurs, les régions
internes, tannophiles, se confondent en partie, par la méme raison.
A la périphérie on ne voit plus la membrane de Slavjanski; on en
apercoit les débris, 4 gauche, sous la forme d’un petit cordon
spiralé, un peu épais. Cependant, les limites du follicule sont
bien visibles, car la transition entre les différentes régions du folli-
cule et le tissu conjonctif environnant est trés nette. La portion
restante du follicule, correspondant 4 la granulosa, est occupée
par une substance colorée en gris pale, qui se confond insensible-
ment avec la région externe, pale, des deux pellucides déliques-
centes, dont les limites sont encore visibles, 4 cause de la couronne
de vacuoles qu’y a déterminé le fixateur. Dans la substance
pale qui occupe le lieu de la granulosa on ne voit plus de cellules;
nous ne voulons pas dire qu’elles n’y puissent exister, car la
méthode tanno-ferrique n’est pas apropriée pour les mettre en
relief. Cependant, soit par leur réfringence, soit par une légére
teinte gris-pale, on voit d’habitude les noyaux: par exemple,
ils sont visibles dans l’appendice annexé au follicule. Par contre,
on voit dans la granulosa des filaments tannophiles irréguliers,
ici un peu épais, lA trés fins, parfois isolés, d’autres fois liés en
réticulum. Ces filaments n’ont rien d’atypique; on les voit plus
développés et plus définis dans le moment agonique de l’atrésie
des follicules; nous les étudierons plus tard, dans un autre travail.
Outre ces filaments, on voit encore dans la masse pale qui occupe
la place de la granulosa des coagula plus colorés. Ces coagula

508 A. L. SALAZAR

prennent dans certains points la forme de cordon flexueux, dans
d’autres celle de coagula diffus, 4 contours estompés; dans d’autres
régions encore on les voit présenter un aspect réticulé, car les
masses en question y sont criblées de vacuoles.

Nous ne savons pas ce qui représente la masse pale qui occupe
la place de la granulosa. Elle peut résulter de la déliquescence
des cellules de la granulosa ou de l’épanchement au dehors de la
substance qui constitue la région externe, pdle, des pellucides.
Cette derniére hypothése semble d’abord la plus vraisemblable;
mais si l’on remarque que les limites externes des pellucides sont
encore marqués, grace 4 la couronne de vacuoles artificiels; que
ces vacuoles ne sont pas visibles dans la masse grisAtre qui occupe
la granulosa; qu’il n’existe dans la zone pdle des follicules les
filaments fins caractéristiques de la granulosa agonique (excepté
& gauche, entre les deux pellucides, ot les limites ne sont pas
nets), on penche surtout vers la premiére hypothéses. L’identité
de coloration ne suffit pas d’ailleurs pour établir Videntité de
substance. I] se pourrait encore que ces deux hypothéses fus-
sent réelles, les deux causes ayant determiné la formation de la
masse pale. Les masses plus tannophiles, qu’on voit dans la
figure et que nous avons décrites plus haut, représentent les
fragments d’un cordon tannophile. La forme en cordon, comme
nous l’avons dit, est encore visible en certains points; dans d’autres
on ne la reconnait plus. La tannophilie de ces fragments est
beaucoup moins intense que celle des cordons que nous avons
décrits dans les autres follicules. Il semble, en résumé, qu’il
s’agit soit de petits cordons, soit d’un cordon fragmenté, en
deliquescence.

Le follicule n’aurait, en résumé, rien de bien particulier, si
n’était pas la disposition extrémement bizarre qu’on voit dans
la base de l’appendice.

Comme le montre la figure 3, un long cordon trés epais, trés
tannophile, occupe cette région. I] décrit constamment des
flexuosités trés serrés, tout en serpentant autour de la région ot
lappendice s’implante sur le follicule, région qu’il cache. En-
suite, conservant toujours sa coloration intensivement noire, il
diminue graduellement d’épaisseur et rampeautour del’appendice,

FOLLICULES DE GRAAF A CORDON TANNOPHILE 509

toujours ondulant et serpentant: puis il disparait avec l’appen-
dice, dont nous n’avons pu voir l’extrémité, comme nous l’avons
dit. Dans la base de l’appendice le cordon, toujours flexueux,
ondule 4 la superficie du follicule. Une partie du cordon semble
logé dans l’intérieur du follicule et dans la base de l’appendice,
une autre partie semble extérieure. Un peu au dessus, dans la
région oti l’appendice sort du follicule, en s’amincissant, tout le
cordon semble extérieur 4 l’appendice; plus loin, quand le cordon,
plus mince, rampe adossé 4 l’appendice, il est toujours extérieur;
s'il existe, 4 la base de l’appendice, des troncons qui semblent
logés dans l’intérieur du follicule et dans la base de l’appendice,
il faut avouer que l’absence de membrane de Slavjanski et l’as-
pect embrouillé de la préparation dans cette région laissent l’esprit
dans le doute. Quoiqu’il en soit, la plus grande partie du cordon,
du moins, est nettement extérieure; cela fait penser tout de suite
que ce cordon n’est que la membrane de Slavjanski et la mem-
brane propre de l’appendice devenues hypertrophiques. En
effet, nous avons démontré A l’aide du tannin-fer que les cordons
ovigenes poss¢dent une membrane propre,’ ce que Paladino avait
toujours nié; cette membrane existe également dans les reliquats
des cordons, soit quw’ils restent libres, soit qu’ils demeurent an-
nexés au follicules. Nous avons montré que ces membranes
propres peuvent s’hypertrophier et se transformer en cordons
flexueux tannophiles dans l’atrésie des cordons et de leurs
reliquats. Comme d’habitude il arrive la méme chose pour la
membrane de Slavjanski (qui n’est pas autre chose, comme
nous l’avons montre,* qu’un fragment de la membrane du cordon
ovigene), on comprend aisément que l’aspect représenté dans la
figure 3 puisse étre determiné par l’hypertrophie simultanée de la
membrane du follicule (gonflée dans le voisinage de l’appendice,
disparue dans la portion restante) et de la membrane de l’appen-
dice. Mais cette explication est immédiatement démentie si
l’on observe ce fait déconcertant, 4 savoir, que l’appendice con-
serve intacte sa membrane propre. Dans la figure on peut la
voir dans toute l’extension du long appendice, sous la forme de
deux traits minces qui le limitent des deux cdtés. Ces traits dis-
paraissent ici et 14 sous les flexuosités du cordon, divergent dans

510 A. L. SALAZAR

la région ot lappendice sort du follicule et disparaissent plus
loin. Done, le cordon tannophile ne semble pas provenir de
Vhypertrophie atrésique des membranes limitantes en question;
d’ou' vient-il alors? Est-ce que le cordon, d’abord intérieur, logé
dans la granulosa, a émigré vers l’extérieur? Cette hypothése est
trés peu probable. Est-ce que, graceaux constantesremaniements
de l’ovaire, le follicule en question a été ficelé par un cordon
provenant d’un follicule disparu, le cordon ayant resté, sous
forme de débris, dans le strome, comme il arrive souvent? L/’in-
timité des rapports existants entre le cordon, l’appendice et le
follicule, rend cette hypothése aussi improbable que les autres.
Faute de mieux, nous reviendrons ainse a la premicre hypothése.
Mais comment harmoniser cette interprétation avec ce fait
paradoxal, la conservation de la membrane dans l’appendice?
On peut le faire de deux mani¢res. En effet, sil’on suppose que la
membrane de Slavjanski a subi l’hypertrophie habituelle dans la
région voisine de l’implantation de !’appendice et que la membrane,
ainse hypertrophiée, rampe, au fur et 4 mesure de sa croissance,
au long de l’appendice, grace 4 une attraction chimio-taxique
quelconque, on aurait l’image représentée dans la figure. Mais
on peut encore admettre que le cordon tannophile provient en
réalité de ’hypertorphie simultanée d’une partie de la membrane
folliculaire, celle qui voisine la base de l’appendice, et de la mem-
brane propre de celui-ci. Car, si l’on suppose que la membrane
propre des cordons ovigénes et de leurs reliquats n’est pas un
élément homogéne, simple, mais une formation complexe, on
peut tout de suite admettre une dissociation atrésique de ses
éléments, les uns subissant l’hypertrophie, les autres non. La
membrane pourrait étre, par exemple, d’origine épithéliale et
conjonctive 4 la fois, mais nous verrons plus loin que cette hypo-
thése est peu admissible. En admettant qu’elle est seulement
d'origine épithéliale, on peut supposer que le produit élaboré se
différentie ensuite, cette différentiation étant l’index soit d’une
structure complexe, soit d’une substance qui se présente avec
des étapes de maturation diverses, progressant vers l’extérieur.
Les choses peuvent méme ¢tre encore plus simples. Car les
traits qui délimitent l’appendice ne représentent peut-étre autre

FOLLICULES DE GRAAF A CORDON TANNOPHILE oLt

chose qu’une sorte d’exoplasme ou de cuticule de la cellule,
normalement caché sous la membrane propre adhérente. Cette
derniére explication nous semble la plus probable, car nous avons
observé des faits semblables dans la membrane des follicules de
De Graaf. On y voit parfois, au début de Vhypertrophie atrésique
de la membrane de Slavjanski, partir de la portion déja épaissie
un cordon plus ou moins long, que ondule 4 périphérie du folli-
cule; ce cordon suit la membrane qui reste encore visible. Pour
rendre plus claire notre idée, nous nous servirons d’une comparai-
son. Dans les coupes colorées par le tannin-fer on ne peut dis-
tinguer la pellucide de la membrane vitelline; dans ces coupes la
pellcuide, ou mieux, certains types de pellucide y apparaissent
sous la forme d’une bande homogéne colorée en noir intense.
Cet anneau noir, par son bord interne, délimite nettement |’oocyte,
de sorte que la membrane vitelline y apparait noyée dans la
tannophilie intense de la pellucide. Si quelque chose d’analogue
existe dans les membranes des cordons ovigénes, de leurs reli-
quats et de la membrane de Slavjanski, le cas de la figure 3 serait
d’une explication plus facile.

B. RAPPORTS DE CE TYPE D’ATRESIE AVEC L’ATRESIE HYDRO-
PIQUE DES CORDONS OVIGENES ET DE LEURS RELIQUATS

Done, quoique l’aspect de ces follicules atrésiés soit tres bizarre,
seule existence du cordon caractérise ces follicules: l’oocyte et
la pellucide présentent des aspects qu’on touve partout dans
les follicules atrésiés. Quelle est l’origine et la signification de ce
cordon? Les faits, que nous avons signalés dans l’atrésie des
cordons ovigénes et de leurs reliquats,! rendent assez facile l’ex-
plication de ces cas. Ces faits, signalés dans une note trop suc-
cinte, seront décrits en détail et largement illustrés dans un
mémoire. Nous rappellerons ici les faits indispensables pour la
discussion du cas présent: nous renvoyons le lecteur, pour la
description compléte au mémoire illustré, qui paraitra prochaine-
ment. Dans les interstices entre les cellules des cordons ovigénes
ou de leurs reliquats quand les coupes, fixées dans le liquide de
Bouin, ont été colorées par notre procédé au tannin-fer, on voit
souvent s’accumuler une substance liquide ou pateuse, 4 figure

512 ANS. SALAZAR

de coagulation homogéne et trés tannophile (fig. 4). Cette sub-
stance, en s’accumulant dans les interstices entre les cellules
épithéliales, dissocie et désagrége le cordon ovigéne ou le
reliquat; c’est ce que nous avons appelé l’atrésie hydropique, car
elle consiste dans V’accumulation pour ainsi dire monstrueuse
d’une substance liquide ou semi-liquide dans l’intérieur du reli-
quat ou du cordon ovigéne. Cette hydropisie tannophile est
trés fréquente dans les ovaires du type ovigéne, mais on la ren-
contre dans les ovaires du type folliculaire, atrésique et méme
interstitiel; seulement, dans ces derniers types on ne voit plus
des cordons ovigénes hydropiques mais uniquement des reliquats.
Cette hydropisie est, en effet, la seule forme de dégénérescence
connue des reliquats, grands ou petits (les follicules anovulaires
de Regaud, par exemple).

Or, dans quelques reliquats, et parfois aussi dans les cordons
ovigénes, on ne voit plus un magma hydropique a l’intérieur,
mais un cordon tannophile flexueux, parfaitement analogue 4
ceux que nous venons de décrire (fig. 5). Ce cordon tannophile
se loge dans les interstices entre les cellules du reliquat, tout
comme la substance tannophile hydropique; il ne doit pas étre
confondu avec un cordon également tannophile et flexueux,
qu’on voit parfois 4 la périphérie du reliquat; ce dernier n’est
autre chose que l’hypertrophie atrésique de la membrane du cor-
don ovigéne ou du reliquat. |

Entre les reliquats qui sont en dégénérescence hydropique
typique et ceux qui présentent un cordon tannophile a l’intérieur
il existe toutes les passages. Le cordon est constitué par une
substance plus dense, modelée, présentant une forme définie et
indépendante, tandis que dans l’hydropisie tannophile typique
nous voyons une substance plus fluide, qui ne posséde pas de
forme propre et emprunte celle des cavités et interstices ot elle
se trouve logée. Nous ne voulons pas dire que le cordon tanno-
phile soit une étape plus avancée de l’hydropisie, car, s'il était
ainsi, nous trouverions toujours un cordon tannophile dans les
reliquats atrésiques des ovaires du type interstitiel tandis qu’on
y trouve, au contraire, l’atrésie hydropique typique. La forma-
tion du cordon tannophile représente ainsi simplement une

FOLLICULES DE GRAAF A CORDON TANNOPHILE 5£S

modalité spéciale de l’atrésie hydropique: les deux formations
ont, done, la méme origine et le méme processus génétique. Or,
Vorigine de la substance hydropique n’est pas difficile 4 établir.
Dans les interstices entre les cellules des cordons ovigénes et de
leurs reliquats il existe un liquide qui est homologue du liquor
interstitiel et du liquor de antrum des follicules (fig. 5). Ce
liquor interstitiel des cordons et de leurs reliquats est mis en
évidence par le tannin-fer, qui laisse les cellules en blanc, tout
comme dans le granulosa des follicules de Graaf: seulement la
coloration est ici beaucoup plus faible et plus insconstante. La
_ substance hydropique semble résulter d’une hypertrophie atré-
sique de ce liquor interstitiel; elle est homologue de l’hypertrophie
du liquor interstitiel, qu’on voit souvent, quoique d’une forme
plus diseréte et moins dominante, dans la période post-chromato-
lytique des follicules de De Graaf atrésiés, quand ils sont
colorées par le tannin-fer. De sorte que le cordon tanophile
doit étre considéré ainsi comme une modification atrésique du
liquor interstitiel des reliquats.

Cette modification peut étre interprété de deux fagons: ou il
s’agit d’une altération directe de ce liquor ou d’une altération
indirecte. Dans le premier cas, on doit supposer que le liquor
svaltére grace & un déséquilibre quelconque de la dynamique
métabolique du reliquat; dans le second cas, on peut admettre
que les cellules du reliquat, ayant ségrégué d’abord le liquor
interstitiel quand elles étaient encore normales, produisent en-
suite une nouvelle substance hydropique qui, passant dans les
interstices, s’y accumule et forme tantdt la substance hydropique,
tantot le cordon tannophile; c’est-a-dire, il s’agit dans cette
derniére hypothése d’une altération atrésique de la dynamique
cellulaire. )

Quoiqu’il en soit, nous voyons que l’existence d’un cordon
tannophile n’est nullement exclusive des follicules que nous
venons de décrire. En effet, le cordon tannophile de ces fol-
licules est absolument analogue A celui qui se forme dans les
reliquats épithéliaux. Il présente la méme tannophilie intense,
les mémes flexuosités, la méme localisation. La seule différence,
d’importance nulle, se rapporte A la longueur du cordon, beau-

514 A. L. SALAZAR

coup plus grande dans les follicules que dans les reliquats. Un
fait important 4 propos de cette homologie est le suivant. Nous
avons dit plus haut qu’il existe des ovisaes avec cordon tanno-
phile qui remplit presque tout le follicule, comme dans la figure 1,
d’autres ot! le cordon est plus court, d’autres encore ot il est
constitué par des troncons fragmentés, en partie confondus avee
les magma atrésiques. Ces derniers existent das les ovaires du
type ovigéne et non en tous: jJusqu’ici, nous avons trouvé ces
follicules & cordon long seulement dans l’ovaire caractérisé par
l’xistence d’une grande poussée de cordons ovigénes. Or, c’est
précisément dans ces ovaires qu’on trouve les reliquats avec
cordon tannophile analogue. D’un autre cdté, parmi les folli-
cules 4 cordon ce sont les plus petits qui présentent rélativement
le plus grand cordon (comparez figs. 1 et 2). Ceux qui sont
presque entiérement remplis parle cordon sont encore trés proches
de l’étape primordiale; il est méme possible que quelques folli-
cules tout jeunes prennent, 4 cause du développement du cordon,
des dimensions supérieures 4 celles qui leur sont habituelles.
Tout cela—coexistence dans les ovaries du type ovigéne de
follicules 4 grand cordon avec des reliquats qui présentent dans
leur intérieur un cordon analogue; le plus grand développement
du cordon dans les follicules trés petits—semble indiquer qu’il
existe des rapports entre ce type spécial d’atrésie et l’existence
d’une poussée ovigéne trés active. Il se peut que cette forme
d’atrésie ne soit autre chose qu’une manifestation d’un processus
caractéristique des reliquats. Cette manifestation, normale dans
les reliquats, serait aberrante dans les follicules. Car le nombre
de reliquats 4 cordon est beaucoup plus nombreux que celui
des follicules qui présentent également celui-ci. Ce contraste
est trés net, puisque les reliquats qui restent annexés aux folli-
cules présentent souvent (dans les ovaires ovigénes) le cordon
tannophile, tandis que le follicule ne présente aucun signe de
dégénérescence. Cette manifestation, aberrante dans les folli-
cules, semble étre une sorte de désorientation présentée par un
processus d’habitude orienté dans un sens défini dans les cordons
ovigénes et dans leur reliquats; pour rendre claire notre pensée
nous dirons qu’il s’agit d’une sorte de mouvement des cordons

FOLLICULES DE GRAAF A CORDON TANNOPHILE A Ws)

ovigénes et des reliquats qui s’est propagé par inertie Jusqu’aux
follicules. Ce retentissement, trés marqué quand le follicule
dégénére dés le premier Age, s’estompe plus tard, quand l’ovisac
n’est atteint par l’atrésie qu’aprés avoir parcouru une certaine
portion de sa trajectoire évolutive. I] est méme possible que ce
retentissement, plus ou moins accentué, concoure pour que le
follicule dégénére plus précocement. L’étude de ces influences
sur latrésie des follicules de De Graaf nous semble d’une grande
importance pour expliquer certains types d’atrésie, qui s’écartent
du processus atrésique typique de l’ovaire adulte, qui le type
chromatolytique. D’une maniére générale, les processus atré-
siques embryonnaires, c’est-a-dire ceux qui frappent la premiére
et la deuxiéme prolifération et les follicules de l ovaire embryon-
naire, dont la connaissance est due surtout aux travaux de
Winiwarter et Sainmont, contrastent nettement avec l’atrésie
typique de l’ovaire adulte. Dans l’atrésie embryonnaire nous
voyons l’activité dissociante du conjonctif, la fragmentation et
la dissociation des ovisacs, etc., maiS rien qui rappelle, méme
dans les follicules, les phénomeénes décrits dans ce que nous avons
appelé les périodes pré-chromatolytique, chromatolytique et
post-chromatolytique.4 Mais, si nous étudions un ovaire adulte
du type ovigéne, nous voyons ce contraste s’attenuer. Car nous
voyons, dans l’atrésie des cordons, 4 coté des processus spéci-
fiques, d’autres qui ne le sont pas: des phénoménes de chroma-
tolyse discréte; dans l’atrésie des follicules, 4 cdté des processus
chromatolytiques typiques, nous voyons d’autres qui ne le sont
pas: par exemple, la pénétration et la dissociation conjonctive,
trés atténuée. Nous y voyons encore des processus spécifiques,
comme celui qui caractérise les follicules 4 cordon tannophile,
que nous venons de décrire. De Winiwater et Sainmont® ont
d’ailleurs insisté déja sur que les processus d’atrésie se substituent
lentement les uns aux autres. Or, l’ovaire 4 type ovigéne semble
représenter une étape de transition, placée au début de l’age
adulte. On y voit les derniers vestiges des processus atrésiques
embryonnaires, des processus atrésiques spécifiques et des proc-
essus atrésiques adultes. Cette coexistence semble indiquer
un changement important dans la dynamique des processus

516 A. Li SALAZAR

atrésiques, car dans les ovaires du type ovigéne, oti la poussée
n’est plus en pleine vigueur, et dans ceux qu’ont déja presque
atteint le type folliculaire ot elle est éteinte, on voit disparaitre
entiérement les vestiges des processus atrésiques embryonnaires,
s’atténuer ou disparaitre les processus spécifiques du type ovi-
géne bien caractérisé, 4 poussée ovigene intense, et, par contre,
se généraliser les processus atrésiques habituels du type adulte.
D’un autre cé6té, si nous examinons les processus atrésiques qu’on
voit dans l’ovaire du type folliculaire, du type atrésique et du
type interstitiel, on remarquera que les follicules dégénérent par
atrésie chromatolytique seulement aprés qu’ils ont atteint une
certaine grosseur, les petits follicules, au contraire, dégénérant
en général d’une maniére différente. Ces processus atrésiques
des petits follicules, encore mal étudiés, ne sont peut-étre que
des reliquats de processus dégénératifs caractéristiques des
étapes antécédentes. Tout cela, il faut le dire, est basé sur que
les ovaires du type ovigéne, folliculaire, atrésique, interstitiel,®
représentent |’évolution de l’ovaire comme organe, mais ceci n’est
qu’une hypothése fondée sur la comparaison et les rapports de
ces types; l’évolution de l’ovaire adulte de la Lapine est encore
aujourd’hui absolument inconnue, car les recherches de Wini-
warter® n’ont été encore prolongées, jusqu’a l’dge sénile, par aucun
biologiste. Quoiqu’il en soit, il n’est pas moins vrai que le type
d’atrésie des follicules 4 cordon tannophile doit étre considéré
comme une répercussion des processus atrésiques spécifiques des
cordons ovigénes et de leurs reliquats, dans les ovaires adultes
du type ovigeéne.

Une autre question est la suivante. Le cordon tannophile des
follicules et celui des reliquats, nous le savons déja, sont des
formations analogues; done, ce que nous avons dit & propos de la
formation du cordon dans les reliquats s’applique 4 celui des
follicules. Cependant, il y a, 4 ce propos, un point & discuter,
d’importance capitale. En effet, nous avons attribué la forma-
tion du cordon tannophile 4 une aberration des processus sécré-
toires des cellules épithéliales, soit du reliquat, soit du follicule.
Mais la formation du liquor folliculi aux dépens des cellules de
la granulosa n’est pas encore un fait absolument prouvé. Cette

FOLLICULES DE GRAAF A CORDON TANNOPHILE 517

question a été étudiée par certains auteurs, V. der Stricht, par
exemple, qui n’en ont pas donné des preuves absolument décisives;
elle n’a pas encore été posée pour les reliquats et les cordons
ovigénes, ou, que nous sachions, l’existence d’un liquor interstitiel,
homologue du liquor folliculi, n’avait été pas encore signalée
jusqu’a nos travaux. Nous disons homologie et non identité,
car non seulement le liquor interstitiel des reliquats et celui des
cordons, d’un cété, le liquor primordial* et leurs dérivés, de
Vautre, ne présentent exactement les mémes caractéres, mais
il faut remarquer encore que la formation du follicule primordial
marque pour les cellules de la granulosa un changement de vie,
de destinée et de fonctions qui les écartent, au point de vue
fonctionnel, des cellules des reliquats, dont elles sont cependant
les sceurs. Cette orientation nouvelle de leur vie est due 4
Vapparition de l’oocyte et elle débute en plein cordon ovigéne,
dés que les cellules de ce cordon se sont différenciées en oocyte
et en cellules satellites; tandis que l’absence de cette différentia-
tion déterminera la formation d’un reliquat plus ou moins volu-
mineux, ot les cellules n’ayant plus, 4 ce qu’il semble, de roéle
a remplir, ou ayant un tout autre role actuellement inconnu, ne
se différentient ni se multiplient plus. Elles vivent dans le
reliquat une vie végétative, y évoluent parfois d’une maniére
désorientée et finissent les unes pour se noyer dans les magma
hydropiques qui dissocient les reliquats, les autres pour tomber
dans le tissu conjonctif, ot leur destinée ne peut étre déterminée.
Mais cette différence, A certains égards fondamentale, ne posséde,
au point de vue qui nous occupe, qu’une importance trés secon-
daire; et la question, qui depuis longtemps se débat & propos de
la formation du liquor folliculi, peut étre généralisée au liquor
interstitiel des reliquats.

Or, lapplication de la méthode tanno-ferrique 4 l’étude de
cette question semble précisement fournir des preuves, qui sont
contraires au role élaborateur des cellules épithéliales des reliquats
et des follicules. Car cette méthode colore le liquor primordial
et leurs dérivés (le liquor interstitiel, le liquor de l’antrum, la
substance liquide des Corps de Call et Exner et la pellucide) en
noir intense, mais ne colore aucun enclave dans les cellules

518 A. L. SALAZAR

épithéliales en question. Le liquide séreux qui, d’aprés V. der
Stricht,® est élaboré dans les cellules de la granulosa des ovisacs
de la Chauve-souris et passe ensuite dans les interstices, ne se
colore pas avec le tannin-fer. Done, ce produit séreux, s’il existe,
n’est pas tannophile et change de constitution quand il tombe
dans le liquor interstitiel. Dans l’évolution de la granulosa,
depuis l’étape caractérisée par l’existence d’une seule rangée de
cellules cubiques, étape ot le liquor folliculi se montre par la
premiére fois, Jusqu’a l’age mtr du follicule, jamais des enclaves
tannophiles ne se montrent dans le cytoplasme des cellules
épithéliales du follicule. Mais ce fait n’a, en somme, aucune
importance, car il suffit de penser que la substance élaborée par
la cellule peut changer de constitution quand elle est expulsée.
Les cas de ce genre sont trop fréquents dans les processus de
sécrétion glandulaire pour qu’il faille y insister ici. Par contre,
il existe une série de faits, qui nous portent 4 admettre le role
actif des cellules épithéliales du follicule dans l’élaboration du
liquor. A ce propos, nous ne devons pas envisager les cellules
en question d’une maniére étroite et localisée, mais dans l’en-
semble de leur vie, de leur évolution, de leurs changements et de
leurs manifestations, soit dans le follicule normal, soit dans le
follicule atrésique. Parmi ces faits, la plupart trés connus, nous
rappellerons ceux que nous avons derniérement mis en relief.
Le liquor folliculi se montre, comme la méthode tanno-ferrique
le met en évidence, dés l’étape de follicule primordiale. Il forme
alors ce que nous avons appelé le liquor primordial. Ce liquor
primordial est une sorte de blastéme, d’ou dérivent plus tard le
liquor interstitiel, le liquor de l’antrun, la substance liquide des
Corps de Call et Exner et la pellucide. Or, ces différentiations
progressives sont toujours acompagnées d’un remaniement des
cellules épithélialis qui semblent présider et orienter la différen-
tiation du liquor primordial. C’est ainsi, par exemple, que la
figure de précipitation de la substance liquide des Corps de Call
et Exner change dés que les cellules se sont disposées en rosace
autour du noyau central. En résumé, la vie méme de la cellule
épithéliale du follicule nous fournit un ensemble de faits plus
important pour définir son réle élaborateur que la démonstration

FOLLICULES DE GRAAF A CORDON TANNOPHILE 519

histologique d’une substance endo-cellulaire quelconque en rap-
port immédiat avec ce réle élaborateur. Nous ne voulons,
d’ailleurs, nullement nier que la transsudation provenant des
vaisseaux de la théque ne puisse remplir un role dans l’élabora-
tion du liquor; ce role, qu’il se résume en fournir 4 la cellule de la
eranulosa des des matériaux ou qu’il consiste dans une transsu-
dation interstitielle concourant 4 la formation du liquor, n’exclue
nullement l’activité des cellules épithéliales dans l’élaboration du
liquor. Or, cela étant ainsi, on comprend aisément qu’un
déséquilibre atrésique dans l’activité des cellules en question
puisse produire des produits atypiques. Le cordon tannophile
des follicules, que nous venons de décrire, est un produit de ce
genre, d’aprés notre maniére de voir. Cette aberration de
activité des cellules de la granulosa est une répercussion dans
les follicules de processus dynamiques qui sont normaux dans les
reliquats, ot ils aboutissent 4 la formation des magma tannophiles
dans la dégénérescence hydropique. Nous ne voulons pas dire
que ce mouvement transmis aux follicules et qu’y détermine ces
formes atrésiques rares représente un cas anormal; la disparition
progressive du cordon dans les follicules de plus en plus Agés,
montre, au contraire, qu’il s’agit d’un phénomene systématique.
Il s’agit, en somme, d’aprés nous, d’un cas particulier qu’il faut
intégrer dans un complexus de phénoménes encore mal étudiés,
que nous avons signalés plus haut, 4 savoir: la répercussion
jusqu’aux follicules de phénoménes spécifiques aux poussées Ovi-
genes. Ces phénoménes s’attenuent de plus en plus, au fur et a
mesure que le follicule est frappé d’atrésie dans un Age plus
avancé, donc plus écarté du moment ovigéne; cette atténuation
est acompagnée d’une évolution progressive de l’atrésie vers le
type adulte, chromatolytique. Mais cette répercussion peut
aller plus ou moins loin, et cela devra nous expliquer un cer tain
nombre, du moins, de formes d’atrésie atypiques.

Nous voulons faire remarquer encore un autre fait. La res-
semblance entre les cordons tannophiles des follicules et l’aspect
de la membrane de Slavjanski atrésique est frappante; la méme
ressemblance existe entre le cordon tannophile des reliquats et la
membrane hypertrophiée, qu’on voit parfois autour des reliquats

520 Aor SARAZAR

et qui est homologue de la membrane folliculaire atrésiée.
Parfois méme, dans les reliquats, le cordon tannophile intérieur
se continue avec le cordon extérieur sans aucune séparation.

On voit, en somme, que toutes ces formation atrésiques sont
soeurs, possédent la méme origine et sont dues 4 des processus
histo-dynamiques analogues. Or, sil’on admet que la formation
du cordon tannophile dépend d’une modification atrésique du
role sécrétoire des cellules épithéliales soit du follicule soit du
reliquat, comme nous l’avons avancé plus haut, on doit admettre
également la participation des mémes cellules dans l’hypertrophie
atrésique de la membrane de Slavjanski, phénomene presque
constant dans la période post-chromatolytique et méme dans la
période agonique de presque tous les types d’atrésie.

BIBLIOGRAPHIE

1 A. L. Sanazar 1921 Sur les cordons ovigénes de l’ovaire adulte de la
Lapine; leur atrésie. Compt. Rend. de la Soc. de Biol., T. 84, p. 235.

2 Méthode’ de coloration tanno-ferrique. Compt. Rend. de la Soc. de
Biol., T. 83, p. 1655.

3 Sur le follicule de De Graaf non atrésique de la Lapine. Compt. Rend.
de la Soc. de Biol., T. 83, p. 1. 658.

4 Sur la période chromatolytique de la granulosa atrésique de la Lapine.

Mémoires publiés par la Société Portugaise de Sciences Naturelles.
Série Biologique, no. 2.

5 De WINtwarTER ET SainmMontT 1908-1909 Nouvelles recherches sur l’ovo-
genése et l’organogenése de l’ovaire des Mammiféres (Chat). Arch.
de Biolog., T. 24.

6 De WintwarterR 1900 Recherches sur ror onencse et Vorganogenése de
Vovaire des Mammiféres. Arch. de Biolog., T. 17,

7 O.VAN DER Srricut 1912 Sur le processus de l’excrétion des glandes endo-
crines. Le corps jaune et la glande interstitielle de l’ovaire. Arch.
de Biolog., T.77.

8 A.L.Sauazar Sur I’ évolution de l’ovaire adulte de la Lapine. Compt. Rend.
de la Soc. de Biol., T. 85, no. 30.

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AY ta

PLAQUE 1

EXPLICATION DE FIGURES

1 Follicule petit, avec trés long cordon tannophile. Ovaire du type ovigéne
en pleine poussée. Fixation, liquide de Bouin. Méthode tanno-ferrique. Obj.
zis, oc. 2. Dessin au crayon.

2 Follicule un peu plus grand, avec cordon tannophile. Méme ovaire.
Fixation, liquid de Bouin. Méthode tanno-ferrique. Obj. 75., oc. 2. Dessin
au crayon.

3 Follicules 4 deux odcytes, avec un long appendice annexé (reliquat du
cordon ovigéne). Cordon tannophile dans le follicule et dans l’appendice.
Méme ovaire. Fixation, liquide de Bouin. Méthode tanno-ferrique. Obj.7's,
oc. 2. Dessin au crayon.

4 Follicule anovulaire de Regaud en atrésie hydropique. Ovaire du type
interstitiel. Fixation, liquide de Bouin. Méthode tanno-ferrique. Obj. 7's,
oc.2. Dessin au crayon.

5 Follicule anovulaire petit, avec cordon tannophile. Ovaire du type ovi-
géne. Fixation, liquide de Bouin. Méthode tanno-ferrique. Obj. 4s, oc. 2.
Dessin au crayon.

522

FOLLICULES DE CRAAF A CORDON TANNOPHILE PLAQUE 1

A. L. SALAZAR

SUBJECT AND AUTHOR INDEX

LLEN, EDGAR. The oestrous cycle in
GIVE sTNOUBC MS. «..« a/c aadotereraisieiase sisterere eiarets

Amphibia and insects. On certain features
Ofspermatocenesis Ii: one sesce cesses est
Anuran embryos. The development of the
anterior lymphatics and lymph hearts in. .

ACTERIA and chloroplasts. On the

nature of mitochondria. ITI. The demon-
stration of mitochondria by bacteriolog-
ical methods. IV. A comparative study
of the morphogenesis of root-nodule;......

Bacteria. II. Reactions of bacteria to
chemical treatment. On the nature of
mitochondria. I. Observations on mito-
chondria staining methods applied to.....

Baae, Hauser. J. Disturbances in mamma-
lian development produced by radium
EMANALLOI eee ert: «s/o « Cite lelebaeeets

Betuamy, A. W. Differential susceptibility
as a basis for modification and control of
development in the frog. IL. Types of
modification seen in later developmental
RLGRETh hgaaono cos oo aoe VOD AOMbome oo te 0.0 rc

Birds, with special reference to the origin of
the bursa of Fabricius, the formation of a
urodaeal sinus, and the regular occurrence
of a cloacal fenestra. The development of
ClORCAT IN eee et seat ice ss cease neler

Bowen, Ropert H. On certain features of
spermatogenesis in amphibia and insects. .

Boypren, Epwarp A. The development of
the cloaca in birds, with special reference
to the origin of the bursa of Fabricius, the
formation of a urodaeal sinus, and the
regular occurrence of a cloacal fenestra. ....

Bronchiole. The terminals of the human...

Bursa of Fabricius, the formation of a urodaeal
sinus, and the regular occurrence of a
cloacal fenestra. The development of
the cloaca in birds, with special reference
(nls Cutan Or ANiess saeco heoaodoeeasogoo 7c

See ere loop in the chick. The forma-
in} OM aC hoade Susedo dos caneoee sueeae tric

Cavity, including the germinal epithelium of
the ovary, to vital dyes. The reaction of

the cells lining the peritoneal............. 3

Cells lining the peritoneal cavity, including
the germinal epithelium of the ovary, to
vital dyes. The reaction of the..........

Cells of the guinea-pig. The reticular material
as an indicator of physiologic reversal in
secretory polarity in the thyroid..........

Chick. The formation of the cardiac loop in
UL CES Sea Uta COCO E aD Op ao ORG ECU OTES

Chloroplasts. On the nature of mitochondria.
III. The demonstration of mitochondria
by bacteriological methods. IV. A com-
parative study of the morphogenesis of
root-nodule bacteria and

Cloaca in birds, with special reference to the
origin of the bursa of Fabricius, the
formation of a urodaeal sinus, and the
regular occurrence of a cloacal fenestra.

297

203

133

473

373

The development of the.................. 163

Cloacal fenestra. The development of the
cloaca in birds, with special reference to
the origin of the bursa of Fabricius, the
formation of a urodaeal sinus, and the
regular occurrence of a...................:

Cowpry, E. V. The reticular material as an
indicator of physiologic reversal in secre-
tory polarity in the thyroid cells of the
PUN CAH DIU e ee ee raieelevericieveteiets 25

Cultures. Endothelium in tissue............ 39

CunniINcHAM, R.S. The reaction of the cells
lining the peritoneal cavity, including the
germinal epithelium of the ovary, to
vital dyes

Cycle. Changes in the vaginal epithelium of
the guinea-pig during the oestrous........

Cycle inthe mouse. Theoestrous............

163

D ‘ATRESIE des follicules de DeGraaf (lap-
ine), révélée par la méthode tannofer-
rique. Sur une forme particuliére.......

DeGraaf (lapine), révélée par la méthode
tannoferrique. Sur une forme particu-
liére d’atrésie des follicules de............

Development in the frog. IL. Types of modi-
fication seen in later developmental stages.
Differential susceptibility as a basis for
modification and control of...............

Development of the anterior lymphatics and
lymph hearts in anuran embryos. The.. 61

Development of the cloaca in birds, with
special reference to the origin of the bursa
of Fabricius, the formation of a urodaeal
sinus, and the regular occurrence of a
cloacal fenestracme seston te ieisie'scsoicis sins eterale

Development of the saccus endolymphaticus

503

503

473

163

in Rana temporaria Linné. The.......... 231
Development produced by radium emanation.
Disturbances in mammalian.............. 133

Disturbances in mammalian development
produced by radium emanation..........
Dyes. The reaction of the cells lining the
peritoneal cavity, including the germinal
epithelium of the ovary, to vital.........

133

399

MANATION. Disturbances in mamma-
lian development produced by radium.. 133

Embryos. The development of the anterior
lymphatics and lymph heartsinanuran.. 61
Endolymphaticus in Rana temporaria Linné.
The development of the saccus........... 231
Endothelium in tissue cultures............... 39
Epithelium of the guinea-pig during the
oestrous cycle. Changes in the vaginal... 429
Epithelium of the ovary, to vital dyes. The
reaction of the cells lining the peritoneal
cavity, including the germinal............ 399

ABRICIUS, the formation of a urodaeal

sinus, and the regular occurrence of a
cloacal fenestra. The development of the
cloaca in birds, with special reference to
the origin of the bursa of.................

526 INDEX

Fenestra. The development of the cloaca in

birds, with special reference to the origin

of the bursa of Fabricius, the formation of

a urodaeal sinus, and the regular oceur-
TENCE Of a CLOACAN cn a. arctan e cictasiorie 163

Follicules de DeGraaf (lapine) révélée par la

méthode tannoferrique. Sur une forme
particuliére d’atrésie des................- 503

Formation of the cardiac loop in the chick.
Caters iG aot aoc aheceons ao aan 373

Frog. II. Types of modification seen in later

developmental stages. Differential sus-

ceptibility as a basis for modification and
control of development inthe............. 473

ERMINAL epithelium of the ovary, to
vital dyes. The reaction of the cells lin-
ing the peritoneal cavity, including the .. 399

Guinea-pig during the oestrous cycle.
Changes in the vaginal epithelium of the. . 429

Guinea-pig. The reticular material as an
indicator of physiologic reversal in secre-
tory polarity in the thyroid cellsofthe.... 25

He inanuranembryos. The devel-

opment of the anterior lymphatics and

Ino biee aes. osc aati aisle spine rietete
Human bronchiole. The terminals of the.... 267

NSECTS. On certain features of sperma-
togenesis in amphibiaand..............

ee OTTO F. The develop-
ment of the anterior lymphatics and
lymph hearts in anuran embryos...... 61

APINE), révélée par la méthode tanno-
ferrique. Sur une forme particuliére
d’atrésie des follicules de DeGraaf.... 503

Lewis, WARREN H. Endothelium in tissue
CUIEUTES Hee te een ce as piste eictenetesenst =

Loop in the chick. The formation of the
CATCIAC IRE Cent She ALAS. Sats 20. cease: 373

Lymphatics and lymph hearts in anuran
embryos. The development of the
Pinto) adr Laan SER Roan et et Cen eouErteS 61

Lymph hearts in anuran embryos. The
develne a of the anterior lymphatics ‘A
ry its HERA aE ie ote iat. s ain anos oh

AMMALIAN development produced by
radium emanation. Disturbancesin.. 133

Material as an indicator of physiologic reversal
in secretory polarity in the thyroid cells
of the guinea-pig. ‘The reticular......... 25
Méthode tannoferrique. Sur une forme
particuliére d’atrésie des follicules de
DeGraaf (lapine), révélée par la........... 503
Methods applied to bacteria. II. Reactions
of bacteria to chemical treatment. Onthe
nature of mitochondria. I. Observations
on mitochondria staining................- 208
Mitochondria. I. Observations on mitochon-
dria staining methods applied to bacteria.
Il. Reactions of bacteria to chemical
treatment. Onthe nature of............-
Mitochondria. III. The demonstration of
mitochondria by bacteriological methods.

V. A comparative study of the morpho-
genesis of root-nodule bacteria and chloro-
plasts. On the nature of................. 451

Mouse. The oestrous cycle in the............ 297

Ome US cycle. Changes in the vaginal
epithelium of the guinea-pig during the. 429

Oestrous cycle inthe mouse. The............ 297

Ovary, to vital dyes. The reaction of the cells
lining the peritoneal cavity, including the
germinal epithelium of the..............- 3

ATTEN, BRADLEY M. The formation
of the cardiac loop in the chick.......... 373

Peritoneal cavity, including the germinal
epithelium of the ovary, to vital dyes.
The reaction of the cells lining the........ 399

Physiologie reversal in secretory polarity in
the thyroid cells of the guinea-pig. The
reticular material as an indicator of....... 25

Polarity in the thyroid cells of the guinea-pig.
The reticular material as an indicator of
physiologic reversal in secretory......... 25

ADIUM emanation. Disturbances in
mammalian development produced by.. 1383

Rana temporaria Linné. The development
of the saccus endolymphaticus in......... 231
Reaction of the cells lining the peritoneal
cavity, including the germinal epithelium
of the ovary, to vitaldyes. The......... 399
Reactions of bacteria to chemical treatment.
On the nature of mitochondria. I. Obser-
vations on mitochondria staining methods
applied to bacteria. II.................:. 203
Reticular material as an indicator of physio-
logic reversal in secretory polarity in the
thyroid cells of the guinea-pig. The..... 25
Reversal in secretory polarity in the thyroid
cells of the guinea-pig. The reticular
material as an indicator of physiologic.... 25
Root-nodule bacteria and chloroplasts. On
the nature of mitochondria. III. The
demonstration of mitochondria by bac-
teriological methods. IV. A comparative
study of the morphogenesis of............ 451

S ACCUS endolymphaticus in Rana tempo-
raria Linné. Thedevelopmentofthe.... 231

Sanazar, A. L. Sur une forme particuliére
d’atrésie des follicules de DeGraaf (lapine),
révélée par la méthode tannoferrique..... 503

Secretory polarity in the thyroid cells of the
guinea-pig. The reticular material as an
indicator of physiologic reversal in........

Seti, Raymonp M. Changes in the vaginal
epithelium of the guinea-pig during the
oestroupicy cles .eeiaerneue pee ck teen 429

Sinus, and the regular occurrence of a cloacal
fenestra. ‘The development of the cloaca
in birds, with special reference to the
origin of the bursa of Fabricius, the forma-

fionlomarurodaedl,sy.ma. seeker tee 163
Spermatogenesis inamphibiaand insects. On
cCentain features OL so--o ene = eee ele 1

Staining methods applied to bacteria. II.
Reactions of bacteria to chemical treat-
ment. On the nature of mitochondria.

I. Observations on mitochondria......... 203

Susceptibility as a basis for modification and
coutrol of development in the frog.
II. Types of modification seen in later
developmental stages. Differential...... 473

fee ee Sur une forme par-
ticuliére d’atrésie des follicules de De-
Graaf Uapine), révélée par la méthode.... 508

Thyroid cells of the guinea-pig. The reticu-
lar material as an indicator of physiologic
reversal in secretory polarity in the....... 25
Tissue cultures. Endothelium in............ 39

RODAEAL sinus, and the regular oceur-
rence of a leoacal fenestra. The develop-
ment of the cloaca in birds, with special
reference to the origin of the bursa of
Fabricius, the formation of............+++ 163

INDEX

\ eee epithelium of the guinea-pig
during theoestrouscycle. Changesinthe 429

Vital dyes. The reaction of the cells lining
the peritonea] cavity, including the
germinal epithelium of the ovary, to...... 399

ALLIN,IVANE. Onthenature of mito-
chondria. I. Observations on mito-

chondria staining methods applied to
bacteria. II. Reactions of bacteria to
chemical treatment... 2. ce. cericceeeenis 203

527

Wauuin, Ivan E. On the nature of mito-
chondria. III. The demonstration of
mitochondria by bacteriological methods.
IV. A comparative study of the morpho-
genesis of root-nodule bacteria and chloro-
PAI h ane spon separ Ade Gnarn SOU MOOD OR MOOGO 451

Wuiresipr, Bratrice. The development of

the saccus endolymphaticus m Rana

GeMIporaria: WINE) eer kesnic-trelr velo tenes 321
Witton, Herpert G. The terminals of the

Imuman) bronchiolers,. .eetesmende eee ete On

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